Use of vibrational spectroscopy for dna content inspection

ABSTRACT

This disclosure concerns a cytometry system including a handling system that enables presentation of single cells to at least one laser source. The laser source is configured to deliver light to a cell within the cells in order to induce bond vibrations in the cellular DNA. The system further includes a detection facility that detects the signature of the bond vibrations, wherein the bond vibration signature is used to determine the folding or packing of the DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of the following U.S. patentapplication, which is incorporated by reference in its entirety: U.S.Non-Provisional patent application Ser. No. 13/447,511, filed Apr. 16,2012.

U.S. Non-Provisional patent application Ser. No. 13/447,511, filed Apr.16, 2012 is a continuation of U.S. Non-Provisional patent applicationSer. No. 13/298,148, filed Nov. 16, 2011, now U.S. Pat. No. 8,941,062,issued Jan. 27, 2015, which claims the benefit of the followingprovisional applications, each of which is hereby incorporated byreference in its entirety:

U.S. Provisional Application No. 61/456,997, filed Nov. 16, 2010;

U.S. Provisional Application No. 61/464,775, filed Mar. 9, 2011;

U.S. Provisional Application No. 61/516,623, filed Apr. 5, 2011;

U.S. Provisional Application No. 61/519,567, filed May 25, 2011;

U.S. Provisional Application No. 61/571,051, filed Jun. 20, 2011;

U.S. Provisional Application No. 61/575,799, filed Aug. 29, 2011; and

U.S. Provisional Application No. 61/628,259, filed Oct. 27, 2011.

All of the above-identified applications are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Field: This document relates generally to cellular measurements based onmid-infrared absorption measurements and particularly, but not by way oflimitation, cellular measurements based on mid-infrared absorptionmeasurements using quantum cascade lasers (QCLs)-based architecture forinfrared activated cell sorting (IRACS).

Identification, classification and sorting of cells, in particular livecells, is a subject of considerable research and commercial interest.Most recently systems for sorting stem cells have been an area ofparticular focus. For example, methods for separating cancerous fromnon-cancerous cells have been demonstrated. For another example, thereis an established market for cell sorting for gender offspring selectionby identification and selection of X- or Y-bearing spermatozoa.

There is currently no safe and accurate method for cell sorting. Themost advanced technology uses fluorescence-activated cell sorting(FACS), where living cells are incubated in a fluorescent DNA-attachingdye, exposed to a high-intensity, high-energy UV laser beam, and sortedaccording to observed fluorescence. There are two major disadvantages tothis method applied to certain cells, including low accuracy and safetyconcerns. For example, in sperm cell sorting, the FACS process is ableto achieve 88% X-enrichment and only 72% Y-enrichment, even at very lowsort rates (20-30 per second output). High scattering at UV and visiblewavelengths is a major factor.

In sperm cell sorting, the FACS process has been shown to causechromosomal damage in sperm cells as a result of the dyes used, and as aresult of exposure to high intensity 355 nm laser light.

The use of optical methods to identify and classify cells has manypotential advantages such as speed, selectivity/specificity, and theirnon-invasive nature. As a result, a number of methods have beendemonstrated in which light is used to interrogate cells and determinecritical information. One such method is the use of fluorescent markers,which are chemicals that bind to specific structures or compounds withinthe target cells and are introduced into the mixture of cells. Themixture is subsequently rinsed to remove excess fluorescent markers andthe cells are exposed to intense UV or other short-wavelength radiationin order to “read out” relevant quantities and classify the cell. Thechemical markers provide good specificity. However, these chemicalmarkers may damage or later the function of the target cells, which isparticularly disadvantageous for live cell sorting. In testing, dyesused as markers for DNA, for example, have resulted in chromosomaldamage. Further, the intense UV or visible light used to read the levelof marker in the cell may damage the cell, in particular, DNA damageresults from exposure to high-energy UV or visible photons. Also,because of the wavelengths used in so called fluorescence activated cellsorting (FACS) systems, quantitative measurements (rather than yes/nomeasurements for a particular antibody) are made very difficult, becauseboth the illuminating wavelength and the emitted fluorescence arescattered and absorbed by cellular components. This means that cellorientation becomes an important factor in accurate measurement, and candramatically reduce the effectiveness of the system. For example, spermcell sorts for X- and Y-carrying sperm, which measure the differentialin DNA between cells, require very specific orientation (only 10% ofcells typically meet the orientation criteria), and still provideaccuracy only in the 70-90% range for humans.

Another method to interrogate cells and determine critical informationis Raman spectroscopy. In Raman Spectroscopy, cells are exposed tointense visible or near infrared (NIR) light. This light is absorbed asa result of molecular bond vibrations within the cellular structure.Secondary emission of photons at slightly different wavelengths occurs,according to Stokes and anti-Stokes energy shifts. Measurement of thesewavelengths allows the chemical composition of the cell to be measured.With Raman spectroscopy, the individual photon energy is generally lowerthan that used for fluorescent markers, however, the net energy absorbedcan be very high and unsafe for live cells. Raman scattering is anextremely weak process: typically only 1 in 10̂10 incident photons giverise to a Raman-shifted photon, thus requiring long exposure times togenerate sufficient shifted protons for accurate measurement. WhileRaman may not be suitable for high-volume live cell sorting, it can beuse din conjunction with other methodologies described herein. Highersensitivity methods such as coherent anti-Stokes Raman scattering (CARS)are being developed which may enable high-throughput screening.

One significant drawback of mid-IR spectroscopy is the strong absorptionby water over much of the “chemical fingerprint” range. This hasstrongly limited the application of Fourier Transform InfraredSpectroscopy (FTIR) techniques to applications involving liquid (andtherefore most live cell applications) where long integration times areallowable—so sufficient light may be gathered to increasesignal-to-noise ratio and therefore the accuracy of the measurement. Thelack of availability of high-intensity, low etendue sources limit thecombination of optical path lengths and short integration times that maybe applied. In addition, because of the extended nature of thetraditional sources used in FTIR, sampling small areas (on the order ofthe size of a single cell) using apertures further decimates the amountof optical power available to the system.

One approach to enabling liquid or solid-state measurements in themid-IR is to use surface techniques. A popular method is the use of anattenuated total reflection (ATR) prism that is positioned directly incontact with the substance of interest (sometimes using high pressure inthe case of solid samples). Mid-IR light penetrates from the prism up toseveral microns into the sample, and attenuates the internal reflectionaccording to its wavelength-dependent absorption characteristics.

Another method which was more recently developed is the use of plasmonicsurfaces which typically consist of conductive layers patterned toproduce resonances at specific wavelengths; at these resonances, thereis coupling into substances places on top of the layer, and again,absorption at a specific wavelength may be measured with good signal.Again, however, the coupling into the substance of interest is veryshallow, typically restricted to microns.

Furthermore, one of the problems raised in mid-IR microspectroscopy isthat of scattering. Mie scattering is dominant when the particles in thepath are on the order of the interrogating wavelength. The magnitude andangle of scattering is determined by size of particles and index ofparticles relative to the medium. Problems encountered center about themeasurement of high-index cells using FTIR. Scattered light that is notcaptured by the instrument is misinterpreted as absorption, and resultsin artefacts in the Fourier-inverted spectrum. Some of the causes orpromoters of this scattering loss include: 1) Measurement of cells inair, rather than in a water solution. This causes additional indexmismatch between the medium and cells, dramatically raising scatteringefficiency and angles; 2) Measurement of absorption peaks at highwavenumbers (short wavelengths) where scattering efficiency is higher;3) Insufficient capture angle on the instrument. Typically the captureangle on these instruments is identical to the input angle, not allowingfor light scattered outside of the delivered IR beam angle; and 4)Transflection or other surface-based measurements. These configurationsmay lead to additional artefacts in conjunction with Mie scatteringeffects.

SUMMARY OF THE INVENTION

In embodiments of the present invention, a system and method ofcytometry may include presenting a single sperm cell to at least onelaser source configured to deliver light to the sperm cell in order toinduce bond vibrations in the sperm cell DNA, and detecting thesignature of the bond vibrations. The bond vibration signature is usedto calculate a DNA content carried by the sperm cell which is used toidentify the sperm cell as carrying an X-chromosome or Y-chromosome.Another system and method may include flowing cells past at least oneQCL source one-by-one using a fluid handling system, delivering QCLlight to a single cell to induce resonant mid-IR absorption by one ormore analytes of the cell, and detecting, using a mid-infrared detectionfacility, the transmitted mid-infrared wavelength light, wherein thetransmitted mid-infrared wavelength light is used to identify a cellcharacteristic.

These and other systems, methods, objects, features, and advantages ofthe present invention will be apparent to those skilled in the art fromthe following detailed description of the preferred embodiment and thedrawings. All documents mentioned herein are hereby incorporated intheir entirety by reference.

BRIEF DESCRIPTION OF THE FIGURES

The invention and the following detailed description of certainembodiments thereof may be understood by reference to the followingfigures:

FIG. 1 illustrates the present invention built in a form similar to aflow cytometer.

FIG. 2 shows a potential configuration of laser source to interrogate asample stream, in either flow cytometer configuration as shown in FIG.1.

FIG. 3 illustrates a simplified example of mid-infrared spectra for aflow such as those described in FIG. 1 and FIG. 2.

FIG. 4 shows an example configuration of a system interrogating cells ina flow, which is shown in cross-section.

FIG. 5 shows an embodiment of the present invention, in which the flowand cells 120 and 122 are measured from multiple angles.

FIG. 6 illustrates simplified detector signal, corresponding to thesample spectra shown in FIG. 3.

FIG. 7 shows another embodiment of the present invention, where cellsare measured in a dry state.

FIG. 8 shows the application of the present invention embodied using amicrofluidic-type cell sorting system.

FIG. 9 shows a portion of a very basic embodiment of the presentinvention which is a microfluidic system for live cell measurements.

FIG. 10a shows detail of an embodiment of a measurement region in amicrofluidic channel.

FIG. 10b shows the same example as 10 a in cross-section.

FIG. 10c shows a cross-section of an alternative embodiment that may usea reflective measurement for the mid-IR light.

FIG. 11 shows an embodiment of the invention where the microfluidics andQCL-based spectral measurement system may be combined with a moreconventional fluorescence-based measurement system.

FIG. 12 shows an exemplary embodiment of the present invention where themicrofluidic subsystem includes a cell-sorting fluidic switch.

FIG. 13a shows an alternative microfluidic-based embodiment of thepresent invention, where a series of microwells may be integrated into amicrofluidic flow channel/chamber.

FIG. 13b shows how the wells may be then scanned using mid-IR light fromone or more QCLs, the scanning may be accomplished by translating themicrofluidic chip, or the laser leading mechanism.

FIG. 14 depicts another embodiment in which the microfluidic chamber maybe 2-dimensional.

FIG. 15 shows an alternate embodiment of the mid-IR optics combined witha microfluidic channel.

FIG. 16 shows a system for the growth and purification of cells based onthe present invention.

FIGS. 17a and 17b shows two potential configurations for QCLs and mid-IRdetectors in the present invention.

FIG. 18 shows an embodiment of the present invention where it may beused to sort live sperm cells for the purpose of pre-fertilization sexselection.

FIG. 19a shows a cross-section of a fluid stream oriented to carry cellsthrough a measurement volume such that flow is into or out of plane ofpaper in this case.

FIG. 19b shows the same configuration with two example cells in theflow.

FIG. 20a illustrates one configuration of the present invention thatminimizes the effect of cell position in the flow that results fromwater absorption.

FIG. 20b illustrates the rectangular channel with two hypothetical cellpositions in the measurement volume, a well-centered cell and anoff-axis cell.

FIG. 21 shows an embodiment of the present invention implemented in astandard flow cytometry system, in this case fitted for mid-IR cellmeasurements based on QCLs.

FIG. 22 shows the present invention embodied in an architecture thatallows cell position in a flow to be measured accurately, in order tocompensate for position-dependent variations in mid-IR absorptionsignal.

FIG. 23a shows a sheath flow surrounding the core flow being illuminatedusing mid-IR light from QCL(s).

FIG. 23b shows the same flow with a cell in the measurement volume.

FIG. 24 depicts another embodiment of the present invention in whichmultiple mid-IR beams may be used to interrogate the measurement volume.

FIG. 25a shows a tool that may accept microfluidic chips that may bepre-loaded by user with the cell sample to be measured and/or sorted.

FIG. 25b shows another configuration of the tool described herein.

FIG. 26 shows example displays of DNA content shown to the user of thetool.

FIG. 27 shows an example of a single-unit disposable measurement unit,consisting of a microfluidic chip with plastic carrier that may be usedin a system.

FIG. 28 shows an alternative configuration, where a potentially plasticcarrier may be used together with one or more microfluidic chips, wherethe chip may be a separate piece from the carrier.

FIG. 29 shows some example formats for microfluidic chips that may beused in the present invention.

FIG. 30 shows additional example configurations of microfluidic chipsfor use in the present invention.

FIG. 31 shows the example construction of a microfluidic chip for use inthe present invention.

FIG. 32 shows the detail of an example embodiment of a microfluidicchannel including a measurement volume and a pressure-actuated cellswitch.

FIG. 33 illustrates an embodiment of the optical interrogation systemused in the present invention.

FIG. 34 shows an example of the present invention using coherentanti-stokes Raman spectroscopy (CARS) to measure vibrational bondfingerprints in cells at high speed.

FIG. 35 shows a configuration of a QCL with components to reducecoherence length, so as to minimize spatial dependence in the readout ofcell spectral measurements.

FIG. 36 shows an example of the display/user interface when the tool maybe used for cell sorting.

FIG. 37 shows a sample embodiment of the present invention that mayinclude elements to minimize spot size on the sample, and to rejectscattered light from the sample.

FIG. 38 shows several sample configurations used by others that maysuffer from surface effects that the present invention is specificallydesigned to avoid.

FIG. 39 shows an example embodiment of a microfluidic channel describedin the present invention.

FIG. 40 shows how a microfluidic cavity may be optimized to reduceresonant optical effects, such that as much position dependence aspossible is taken out of the QCL-based fluidic measurement system.

FIG. 41 shows an embodiment of the present invention based on aVernier-tuned external cavity quantum cascade laser.

FIG. 42 further illustrates the Vernier tuning mechanism proposed forhigh-speed mid-IR liquid spectroscopy as part of the present invention.

FIG. 43 shows the examples of multiple wavelengths used in the presentinvention.

FIG. 44 shows another example embodiment of the present invention.

FIG. 45 shows a schematic example of a molecular absorption peak in themid-IR.

FIG. 46 shows a generalized system diagram for measurement of mid-IRabsorption of a particle of cell, including but not limited to livingcells.

FIG. 47 shows one approach to remedying the scattering problem.

FIG. 48 shows an alternative architecture where scattered light ismeasured directly.

FIG. 49 shows an embodiment that has the potential to use QCLs tomeasure particle size and chemical concentration through scattered lightonly.

FIG. 50a depicts shows a simple flow architecture where the fluid to bemeasured flows through a channel.

FIG. 50b depicts a fluid-within-fluid flow.

FIG. 50c depicts an example of a flow where the core has been brokeninto droplets.

FIG. 51a shows an example of scattering efficiency (Qs) of a volume suchas a droplet, as a function of wavelength (lambda).

FIG. 51b shows scattering efficiency as a function of wavelength

FIG. 52a shows a fluidic configuration where a droplet or flow isconfined within a 2D channel or manifold.

FIG. 52b shows a fluidic system where droplets or a core flow iscentered within a larger, 3D core flow.

FIGS. 53a-c show a representative example of a system measuring dropletsor flows using QCL-originated mid-IR beams.

FIG. 54a shows a case where a biological cell (inner circle) iscontained within a droplet, which is contained within an emulsion.

FIG. 54b shows a different technique, illustrate here through the use ofa droplet in an emulsion.

FIG. 55 shows an example of a droplet-based system where dropletscontain biological cells.

FIG. 56 shows a configuration where a solid sample with very sparseparticles of interest.

FIGS. 57a-c show flow and particle configurations that could be used toenhance resonant optical interference measurements in the mid-IR.

FIG. 58 shows one method by which cells or particles could be measuredand sorted.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

The invention described herein presents a novel approach to cellularmeasurements based on mid-infrared absorption measurements. Mid-IRquantum cascade lasers (QCLs) have the potential to focus sufficientenergy onto a single cell to make an accurate, high-speed measurement.QCLs have recently been commercialized by multiple vendors. They arebuilt using the same processes and packaging as high-volume telecomlasers. QCLs offer several advantages over traditional mid-IR sourcessuch as delivering very high spectral power density, delivering veryhigh spatial or angular power density, among other advantages. Thisallows QCLs to put 10,000,000× more effective mid-IR power onto a singlecell than traditional mid-IR sources. This disclosure seeks to describecertain applications enabled by QCLs, such as label-free detectionwithout dyes or labels that can alter or damage cells, measurementsusing mid-IR illumination 25× less energetic than that used in FACS,eliminating photon damage, and high-throughput (>1000 cells/second)capability.

Embodiments of the present disclosure include an Infrared-Activated CellSorting (IRACS). The steps in an IRACS system may include prepare thecell sample (such as by centrifugation, etc.); for each cell flowingthrough: illuminating the cell with mid-IR laser(s), measuring thetransmission of mid-IR wavelength(s), and sorting cells by transmissionlevels. The key “input” parameters of the IRACS system model may be:cells per second entering the measurement volume; and spacing betweencells (“duty cycle”). The input parameters may determine the measurementduration or integration time.

Most of the risks at the micro optical level may be addressed by carefuldesign using existing techniques and materials. In this manner many ofthe mid-IR measurement issues may be avoided. The IRACS system mayinclude a microfluidic channel architecture with one or more of thefollowing features. One feature may be top and bottom mid-IRtransmitting windows forming a channel. Potential materials include: Si,Ge, ZnSe, certain polymers. Si, ZnSe (or similar), polymers may becompatible with visible, NIR, or SWIR (500-1600 nm) opticalinterrogation or manipulation, if desired. ZnSe or similar may be usedto permit visible light viewing, interrogation and possible manipulation(laser tweezers, laser-based cell disabling/destruction). Anotherfeature may be antireflection coatings applied to both sides of eachwindow, such as AR coatings designed for air externally, waterinternally. Another feature may be a channel depth tuned to be a gapthat is out of resonance at the mid-IR wavelength(s). For example, achannel depth of approximately 20 microns may be used. Another featuremay be tilting, or wedge-shaped windows to further reduce effects ofreflections. Another feature may be a channel width that exceeds thespot size of the QCL may be so small shifts in the channel with respectto the beam to prevent creation of false signals.

Further, in the present disclosure, several aspects of particularmeasurements which greatly mitigate the effect of Mie scattering by theinterrogated cell may be: 1) The cell is measured in a water medium (asopposed to a dried cell on a window, in air). This may significantlyreduce the index contrast between the medium and cell. The estimatedreal cellular refractive index may be in the range of n_(cell)=1.27-1.30vs n_(H2O)=1.25 at 1100 cm−1, for a cell/medium ratio of 1.02-1.04, anda nuclear refractive index higher than that; 2) DNA signatures are atthe low-wavenumber (long wavelength) end of the vibrational fingerprintregion. Scattering falls off with longer wavelength, so relatively lesseffect is experienced; and 3) Light is captured over a large angle usinga high NA lens; this means even light scattered over moderate angles (10degrees) may be captured and relayed to the detector.

Regarding the issue of cell orientation, scattering is estimated basedon an equivalent spherical volume, and the computer scatteringefficiency is multiplied by the actual cell cross-section that isdependent on cell presentation angle. This may ignore the fact that whencell cross-section is large, path length is relatively short, whichwould in turn reduce scattering effects (since scattering is a result ofthe phase shift and extinction on passage through the sample vs.surrounding medium). To further reduce the effect of cell orientation,the present invention may consider the following approaches: 1) Removingoutliers from the distribution (which may be caused in large part by theorientation issues); 2) Using a measurement wavelength that may not beat the peak absorption level for DNA, or using a less strongly-absorbingDNA band. A high absorption coefficient results in both largerscattering, and also in more orientation/shape dependence instraight-through absorption. At low absorption, path length may bettercompensate for cross-section changes. At high absorption this may beless true; and 3) Using a shorter wavelength (possibly NIR) to moreprecisely determine cell cross-section/orientation, and compensating foror rejecting certain orientations.

Further, to reduce or compensate for the effects of scattering, thepresent disclosure may employ a plurality of solutions such aswavelength optimization, beam angle and capture angle optimization,scatter detection and compensation, scatter based measurement, and thelike.

In an embodiment, the invention uses quantum cascade laser (QCL)components to directly measure absorption spectra of living cells forclassification. Some areas where QCLs are used may includedifferentiating gender in sperm cells based on differences in X- andY-chromosome mass, separation of stem cells from differentiated cellsbased on DNA/RNA mass change, differentiation of healthy and diseasedcells, identification of cell phase of life, and the like.

In an embodiment of a DNA measurement system, the system utilizesoptical absorption into molecular vibration modes specific to the DNAbackbone to accurately measure DNA content of cells as they pass anoptical illumination and readout system in a fluid stream. The systemrequires no staining or labels, or associated incubation process. Thesystem may present a histogram showing cell count vs. DNA quantity percell, and may optionally allow the user to sort out cells with certainquantities of DNA. The system may include reference measures toestablish cell size (such as a Coulter-type electrical impedancemeasurement, or an optical scattering measurement), or other cellularcomponents (such as protein content), which may further help distinguishcell types, and allow identification of cell agglomerates so as toremove them from the data or the sorted stream.

In various embodiments, one or more QCL may be used to measuretransmission at one or more wavelengths through a living cell. Anabsorption by a cell of the one or more wavelengths may indicateconcentrations or mass of constituent components within the cell.Further an absolute or relative level of absorption may be used toclassify or identify the cell type or state. The absorption lines maycorrespond to vibrational modes of molecular bonds in target molecules.

QCLs may be able to directly emit coherent radiation in the mid-infraredrange, covering at least the 3-15 micron wavelength range, such as 3-4microns. The wavelength may coincide with the regions for infraredspectroscopy, and with the wavelengths used in many of the previousspectroscopic work on cells. The potential advantages of usingmid-infrared light to interrogate cells for classification or sortingare numerous, however. They include very specific signatures, low photonenergy, and direct measurement that potentially allow combinations oflow optical power, fast measurement, and/or high precision measurement.Mid-infrared is an important range, since in this range, molecularvibrations may be measured directly through the use of absorptionmeasurements. Essentially the same vibration signals that are measuredusing Raman spectroscopy are measured. Some major advantages of themid-infrared range include that the absorption rate is much higher thanin Raman, resulting in a significantly higher signal per input photon.Additionally, the photon energy used is extremely low compared tovisible-light or even near-infrared measurements; this means no damageto cells or their components from ionization or two-photon absorptionprocesses. Finally, molecular fingerprints in this range have beenextensively characterized for decades using Fourier transform infrared(FTIR) spectroscopy. As opposed to FTIR, which typically uses a “globar”(glowing filament) source which provides very low spectral and arealpower density, QCLs provide much more power on target and on wavelength,allowing much higher signal-to-noise (SNR) ratio and/or throughput.

QCLs may be manufactured in a number of varieties, including Fabry-Perotand Distributed Feedback (DFB) designs, as well as external cavity (EC)designs where wavelength may be set using external devices, and may bebroadly tunable. Advantages of QCLs as a source for mid-infrared lightfor use in microspectroscopy of living cells may include ability todeliver a large amount of optical power in a narrow spectral band,ability to focus mid-infrared light onto a small spot, ability toproduce significant power levels, ability to source QCLs in smallpackages, and the like.

In an embodiment, mid-infrared QCLs that may be used in the presentinvention generally target but are not restricted to the molecular“fingerprint region” of 6-12 microns. In some embodiments, the presentinvention may be applied to the problem of sorting living cells at highspeed in a label-free manner, using relatively small amounts of opticalpower, and using very low-energy photons such as wavelengths around the1000 cm̂−1 range. In various other embodiments, multiple QCL wavelengthsmay be used to measure relative concentrations of one or more substanceswithin cells, and establish a baseline measurement for the mainmeasurement being performed. The QCL wavelengths may be supplemented byvisible, near-infrared or other wavelength measurements to providereference information such as cell location, shape, orientation,scattering, etc. In an embodiment, multiple QCL wavelengths may begenerated through the use of multiple discrete components, singlecomponents generating multiple discrete wavelengths, broadband QCLs inaddition to a filtering technique, tunable QCL components and the like.A mid-infrared source, such as the quantum cascade laser (QCL)—may beintegrated with microfluidic systems for cell transport, presentation tothe measurement system, and optionally, sorting into specificpopulations.

Microfluidic systems are in broad use in biomedical applications,including high-volume commercial applications. Their fabrication and usemay be well-known. Microfluidics may be used to combine, measure, sort,and filter biochemical samples. In some embodiments, the combination ofmicrofluidics with QCLs and mid-IR detectors to achieve accuratemeasurements of live cells may be described. Microfluidics may enablehigher accuracy in such a system. Mid-IR light may be absorbed verystrongly by water. Such a system with small path length (through liquid)may be highly desirable for systems requiring high throughput, lowintegration time, or very high signal-to-noise ratio. Furthermore, itmakes a repeatable, constant path length desirable in order to eliminatefluctuations due to water stream diameter. Microfluidic devices orcircuits, which are fabricated using semiconductor-like processes, mayhave the potential to provide such repeatability.

In addition, microfluidic devices enable closed-loop, compact systemsthat may be more appropriate for systems produced and used in highvolume, which may be one goal of the present invention. Microfluidiccomponents may be fabricated at low cost, and may therefore bedisposable or recyclable and may offer a low-cost way of maintaining aclean system without run-to-run or patient-to-patient contamination.

In some embodiments, the combination of a label-free cellcharacterization system based on mid-IR QCLs may enablesystems-on-a-chip with cell culturing, filtering, detecting, and sortingon a single chip, thereby minimizing inputs and outputs. The ability touse QCLs rather than fluorescent, magnetic or other labels (that must beattached to cells through specific, sometimes laborious procedures thatmay ultimately affect cell viability) multiplies the number ofoperations that may be performed on-chip for specific biomedical or evenindustrial biological applications.

Microfluidic systems of multiple configurations may be used as describedbelow. Importantly, there is a wide range of prior art in microfluidicsthat can be used in conjunction with this system as a whole(QCLs+microfluidics+mid-IR detectors to measure individual cellproperties). Most of these must be modified to be applicable to thepresent invention through the use of substrates (such as top and bottomcaps or wafers that confine the fluid in the microfluidic structures)that transmit mid-IR light generated by the QCLs.

Multiple embodiments of QCL(s) in the system and mid-IR detectors arepossible, and may depend on the application. In an example, one or morebroadly-tunable QCLs may be used to cover a wide mid-IR spectrumcorresponding to multiple molecular fingerprints for researchapplications. Beams may be combined by use of half-mirrors (withaccompanying loss), thin film interference filters or diffractiongratings. Such a configuration may be used to gather a complete spectralsignature for a cell, for instance, where a new QCL-based measurement isbeing developed. Once useful spectral features may have been identified,such a system based on tunable QCLs may be set up with each tunable QCLtuned to a peak, and then cells may be interrogated at higher speed,such as speed that may be used in a clinical system.

Narrowly tunable lasers may be used to interrogate a specific spectralfeature, where derivative with wavelength information is important. Forinstance, rapid scanning over a small range corresponding to peakabsorption of a cell constituent may significantly improve accuracy insome cases where absolute absorption is highly variable due to otherfactors.

Lower-cost, fixed QCL lasers such as distributed feedback, or DFB-QCLsmay be used in systems where the sought-after spectral features are wellknown. In the simplest configuration, a single QCL and detector may beused to measure a chemical concentration within a cell. In the casewhere multiple constituents absorb at the signal wavelength, additionalfixed QCLs may be added to the system to make reference measurements and“back out” the effect of those non-target constituents. For example, RNAand DNA share several absorption peaks, and to make an RNA concentrationmeasurements in a cell, it is most likely necessary to measure twowavelengths and look at the relative absorption levels rather thanmeasure a single absorption peak corresponding to RNA.

A number of configurations may be possible for the mid-IR detectorscorresponding to the QCLs. In other embodiments, a plurality of detectortypes may be used, including mercury cadmium telluride (MCT)photovoltaic detectors, which may be liquid nitrogen cooled,thermoelectric cooler (TEC) cooled, or room temperature. Pyroelectricand other thermal detectors may also be used where cost is an issue. Oneor more detectors may be used in the system. For example, a system withtwo QCLs may use a single detector by using modulation on the QCLs whichare often used in pulsed mode. The signals corresponding to the twoQCLs, combined with the absorption of the sample, may be then measuredby the detector, and may be separated electronically based on themodulation patterns of the QCLs. Alternatively, thin film interferencefilters may be used to separate the two wavelengths and send them toindividual detectors. In any case, the detectors may have passbandfilters mounted in front of them to reject out-of-band mid-IR lightblackbody radiation from the system components, or broadband signalsrelating to the heating or cooling of the cell as it passes through theQCL and possibly visible/N ear Infra Red (NIR) beams.

In another embodiment, multiple QCLs may use completely distinct opticalpaths, either passing through the same sample volume at differentangles, or using altogether separate measurement volumes where a cellpasses through them sequentially. This simplifies the multiplexing ofwavelengths from QCLs, but does not ensure the same measurement is beingmade.

Another detection method that may be used in an embodiment of thepresent invention is photoacoustic detection. Photoacoustic measurementsmay be used with mid-IR including QCL measurements of gasconcentrations, even at very small concentrations. In this method, amid-IR pulse may illuminate a sample. Owing to absorption by specificchemical species, there is some local heating and expansion. Thisexpansion results in a shockwave which may be picked up by acousticsensors. Because the present invention uses closed liquid channels,there is the potential to use “microphones” to pick up absorptionsignals as a cell is interrogated with one or more QCLs. Suchmicrophones may be integrated into one of the wafers forming the top orbottom “cap” of the microfluidic device, or may be attached externallyto the microfluidic structure.

Another potential detection method that may be implemented inembodiments of the present invention is one that may involve measurementof passive (blackbody) emissions from the cell components, as they arestimulated (heated) using a QCL. For example, the cell may beilluminated with one wavelength corresponding to the absorption peaks ofseveral molecules, one of which is of interest, therefore a simpleabsorption measurement would not provide an accurate answer for themolecule of interest. Mid-IR radiation from the cell is then collected,and filtered using a narrow passband mid-IR filter around anotherabsorption (emission) peak for the molecule of interest (or anothermolecule/bond vibration that is tightly coupled, but not directlyaddressed by the input QCL wavelength). The vibrations induced by theprobe QCL wavelength may translate to an increase intemperature/vibrations, and these may be observed at this secondarymid-IR wavelength. This signal will of course be quite small, but may beelectronically filtered with well-known lock-in techniques.

A number of architectures are presented which may be used in the presentinvention to minimize the effect of water absorption and provideaccurate measurements of cellular biochemical components. This is not anexhaustive list, and the present invention will be applicable to otherarchitecture as well. In an embodiment, the present invention consistsof a flow cytometer in which one or more quantum cascade lasers may beused to measure vibrational absorption characteristics of single cellsin the mid-infrared wavelength range.

Living cells may be interrogated at high speed using preparations suchas traditional flow cytometer-type instruments retrofitted with QCL andmid-infrared transparent liquid handling microfluidics components builtfor cell classification and sorting that have been built withmid-infrared transparent components, substrates such as sheets, tapes ordiscs where live cells are distributed over a surface in a thin film andinterrogated by scanning, and the like. In an embodiment, the presentinvention may be used in conjunction with certain chemical or otheroperations in advance of the measurement that accentuate differencesbetween the cells to be sorted. For example, cells may be stimulatedwith temperature, light, fuel, or other stimulant to enhance biochemicalconcentrations that differentiate cells, for example, by differentiallychanging cell metabolism and therefore input or output products.

In an embodiment, the present invention may enable high-speed spermsorting in a label-free manner, and without exposing cells tohigh-energy UV photons. For example, the separation of sperm by X- andY-type is possible using the fact that the X chromosome hassignificantly more DNA content than the Y-chromosome, thereby enlargingthe overall DNA mass of the sperm cell by 2% or more in mammalianspecies. At least one QCL may be used to probe at least one absorptionband specific to DNA. In other embodiments, a plurality of QCLwavelengths may be used to measure other potentially interferingconstituents. The measurements may be made from more than oneorientation to normalize for the asymmetric shape of the sperm cellhead. In various other embodiments, the measurement process may besupplemented using other lasers in the visible or near-infrared bands tomeasure cell orientation, size, position, density and the like, as wellas additional mid-IR based lasers, such as alternately tuned QCL's. Theamount of mid-infrared light transmitted in the DNA band, as normalizedby the other measurements, may be used to compute total mass of DNA inthe sperm cell. As the amount of DNA for X- or Y-bearing cells for agiven species is very consistent, sperm cells may be sorted into X- andY-bearing populations with one or more mechanisms known in the art suchas flow cytometry-type equipment, microfluidics, methods where cells arespread onto larger substrates, cell cuvettes, and the like.

In an embodiment, the present invention may be a system consisting offluidics for delivering live cells one by one to a measurement volume,laser sources which generate beam(s) that may preferentially act on DNAmolecules within the cells, based on specific molecular vibrationfrequencies, detectors which measure the interaction of the sources withthe DNA as the cell passes the measurement volume and signal processingequipment to capture these signals and process them in order to generatean estimate for DNA quantity in a single cell.

In an embodiment, the system may include an interface that displays ahistogram of cell count vs. DNA quantity, a common plot used to analyzea sample of cells.

In an embodiment, the system may include other measurements made on theindividual cells that may further characterize the cell. These mayinclude, but are not limited to electrical impedance measurements in thefluid channel which may allow the system to estimate size orcross-section of the cell, and detect cell agglomerates which mayproduce false DNA readings, optical measurements in the visible or nearinfrared range of scattering or shape which may help determine celltype, and also measure agglomerates or vibrational optical measurementswhich may serve to quantify other biochemical constituents of the cellunder inspection, including but not limited to measuring protein orlipid concentrations to determine the rough size and type of the cell,and detect cell agglomerates.

In an embodiment, the system may include a method of sorting the cellsindividually based on the DNA content calculated, as well as anyreference signals including those described above. This sorting may bedone in one of a number of ways well known to those skilled in the artof cytometry, fluorescence-activated cell sorting, and microfluidics,including but not limited to electrostatic diversion of droplets, fluidpressure diversion of a stream in microfluidics, mechanical actuation ofmeasurement vs. output channels, and laser-based cell trapping/steering.

In an embodiment, the system may use a microfluidic chip on which atleast a source reservoir, output reservoir and measurement channel havebeen patterned. The chip may be a consumable component that is used onceper sample and then disposed or recycled. The core chip may befabricated from a material that is both biocompatible and compatibleoptically with the wavelengths used for the optical vibrational DNAmeasurement. The core chip may be mounted in a plastic carrier withlarge reservoir capacity, the plastic carrier itself may be used withmultiple core chips, where clogging occurs on the core chip and it isdesirable to dispose of these chips instead of performing an automatedunclogging procedure.

In an embodiment, the present invention may be used, and may bespecialized for the purpose of measuring rate of cell division such asculture growth in a sample in accordance with the characteristic thatDNA per cell doubles in the process of division. This disclosure mayprovide for separating X- from Y-bearing sperm cells in accordance withthe characteristic that X-bearing sperm cells may carry more DNA thanY-bearing cells. This disclosure may provide for measuring occurrence ofaneuploidy in a sample of cells, such as for the purpose of detectingcancerous cells or other cells indicative of a genetic disorder. Thisdisclosure may provide for repeatedly measuring samples for the purposeof assessing effects of potential drugs on cells, such as cancer cells.This disclosure may provide for measuring the cell division ratesimultaneously with assessing drug effect. This disclosure may providefor separating potential cancer cells based on an aneuploidy measurementfrom a larger cell culture, where these cells may be rare. Thisdisclosure may provide for identifying and isolating cancer stem cellsfrom tumor tissue for the purpose of characterizing the stem cells. Suchcharacterization may enable further pharmacological study of the stemcells. This disclosure may provide for other applications where cellularDNA is a marker or potential marker for cell type, activity, orpathology.

Further, the present invention also describes a system for measuring thechemical composition/content of particles in the mid-IR using QCLs. Inan embodiment, the present system describes optical system architecturesto mitigate or harness scattering effects for the purpose of makingthese measurements. The optical system architectures minimize,compensate or harness these scattering effects with a minimum number ofwavelengths, allowing high-speed measurements.

In an embodiment, the cell sorting system described in the presentdisclosure may use a QCL wavelength which corresponds to particular bondvibration frequencies. The QCL illuminates the particle/cell, and ifthat molecule is present, the cell and/or analytes within the cell mayabsorb light at that resonant frequency. The remaining light passingthrough the cell may be measured to determine the amount of light thatwas absorbed and therefore the concentration of analyte. For example,DNA quantity may be measured by illuminating the cell with one or morewavelengths, one or more of which are at/near resonant vibrationfrequencies for the DNA molecule. For sperm cells, measuring DNA mayenable determining whether the sperm cell is carrying X or Y chromosomesbecause of the differential in DNA between X and Y (which is one of 23chromosomes). The cell sorting system may comprise two or more QCLs. Atleast one QCL may correspond to the resonant absorption (signalwavelength) for a target analyte. At least one other QCL may correspondto a nearby wavelength to cancel out background noise from otheranalytes and system artifacts.

The other applications of the present invention may include, but not bythe way of limitation, high-speed cell sorting in the separation of stemcells from other cells, including their differentiated derivatives,sorting live from dead cells, DNA content analysis for tumor biology,isolation of key cell populations in tumors, characterization oflymphoma cells, immune cell sorting, and the like.

A processor architecture may use the transmitted or scattered lightdetected by the detection facility to perform a calculation, such as tocalculate a DNA content of a cell. The processor may implement softwareresident on an associated memory or server.

Throughout this Specification, UV light may be in the range of 10 nm to400 nm, NIR light may be in the range of 0.75-1.7 μm, and visible lightmay be in the range of 390 to 750 nm.

Sorting of sperm cells according to the chromosomes they are carryingmay enable a safe, accurate, label- and stain-free pre-fertilizationgender selection method. Certain of the cytometry systems describedbelow enable such a gender selection method.

In an embodiment, a minimally invasive cytometry system may usevibrational spectroscopy for gender selection. The cytometry system mayinclude a handling system that enables presentation of single spermcells to at least one laser source. The at least one laser source may beconfigured to deliver light to the sperm cell in order to induce bondvibrations in the sperm cell DNA. The at least one laser source emits ata wavelength corresponding to the resonant absorption for DNA and atleast one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Thewavelength of the light delivered by the laser source may be greaterthan one third of the diameter of the sperm cell. A detection facilitymay detect the signature of the bond vibrations, which may be used by anassociated or integrated processor to calculate a DNA content carried bythe cell. The calculated DNA content may be used to identify the spermcell as carrying an X-chromosome or Y-chromosome. This identificationmay be done by the associated or integrated processor. The minimallyinvasive cytometry system may also include a sort facility for sortingthe sperm cell according to the identified chromosomes. The cytometrysystem may further include a second light source configured to deliverlight to a sperm cell within the sperm cells in order to induce ascattering signature, wherein the scattering signature is used toidentify a sperm cell characteristic and wherein the characteristiccomprises one or more of size, cell type, cell density, and cellorientation. The second light source may be one or more of a VIS, UV,and NIR laser source and measurements made by the second light sourcemay be used in gating the QCL-based vibrational measurements, in thisembodiment and others described herein. The cytometry system may furtherinclude a cell destruction or immobilization facility, such as a laseremitting at 1.5 microns, that selectively destroys or immobilizes spermcells based on the chromosome they are carrying. In this embodiment andothers described herein, the second light source can be used tosupplement the calculation of the content of the cell that is doneprimarily by the QCL signal (a second signal that can be used in ascatter plot).

In an embodiment, a cytometry method may include presenting a singlesperm cell, using a handling system, to at least one laser source, theat least one laser source configured to deliver light to the sperm cellin order to induce bond vibrations in the sperm cell DNA, and detectingthe signature of the bond vibrations, wherein the bond vibrationsignature is used to calculate a DNA content carried by the sperm cell,wherein the calculated DNA content is used to identify the sperm cell ascarrying an X-chromosome or Y-chromosome.

In another embodiment of a minimally invasive cytometry system forgender selection, the cytometry system may include a handling systemthat presents single sperm cells to at least one quantum cascade laser(QCL) source, the at least one QCL laser source configured to deliverlight to the single sperm cell in order to induce vibrational absorptionby DNA molecules of the sperm cell. A detection facility may detect thetransmitted mid-IR wavelength light, wherein the transmitted mid-IRwavelength light is used to calculate a DNA content carried by the spermcell. The calculation may be done using an associated or integratedprocessor. In this embodiment as well as others described herein, thehandling system may be a manifold/2D array. In this embodiment as wellas others described herein, the handling system may include a carriersubstrate upon which the cells are disposed and the carrier and/or theQCL laser source/detection facility translate with respect to oneanother. In this embodiment as well as others described herein, thehandling system may be a microfluidic flow architecture. Themicrofluidic flow architecture may include multiple microfluidicchannels such that multiple single cell flows may be measuredsimultaneously by the same light source(s). The calculated DNA contentmay be used to identify the sperm cell as carrying X-chromosomes orY-chromosomes. The calculated DNA content may be used to identify ananeuploidy characteristic, such as an extra or missing chromosome or alow DNA count. The cytometry system may further include a second lightsource configured to deliver light to the sperm cell in order to inducea scattering signature, wherein the scattering signature is used toidentify a sperm cell characteristic, and wherein the characteristiccomprises one or more of size, cell type, cell density, and cellorientation. The second light source may be one or more of a VIS, UV,and NIR laser source and measurements made by the second light sourcemay be used in gating the QCL-based vibrational measurements. The atleast one QCL laser source emits at a wavelength corresponding to theresonant absorption for DNA and at least one QCL laser source emits at awavelength used to cancel out a background signal from other analytesand from system artifacts. In this embodiment as well as othersdescribed herein, the cytometry system may further include a sortfacility for sorting the sperm cell according to the identifiedchromosomes. In this embodiment as well as others described herein, whenthe system comprises more than one QCL laser source, the QCL lasersources may be pulsed such that they result in discrete measurements onthe detector, such as alternately pulsed. Indeed, in any of theembodiments described herein, the at least one QCL laser source may bepulsed. In this embodiment as well as others described herein, afacility for electronically separating the transmitted light bywavelength may be included and/or a facility for optically separatingthe transmitted light by wavelength, such as with a dichroic filterand/or a grating. In any of the embodiments described herein, multipledetectors may be used to detect each wavelength. The cytometry systemmay further include a cell destruction or immobilization facility, suchas a laser emitting at 1.5 microns, that selectively terminates ordestroys sperm cells based on the chromosome they are carrying.

In an embodiment of a system using microfluidic architecture for genderselection, a microfluidic architecture may include a fluid handlingsystem that enables a flow of sperm cells past at least one quantumcascade laser (QCL) source, wherein the fluid handling system comprisesa facility to enable a single cell flow in a measurement volume of themicrofluidic architecture. The at least one QCL laser source may beconfigured to deliver light to a sperm cell in the measurement volume ofthe fluid handling system in order to induce resonant absorption by DNAat one or more mid-IR wavelengths. A detection facility detects thetransmitted mid-IR wavelength light, wherein the transmitted mid-IRwavelength light is used to calculate a DNA content of the sperm cellsthat identifies the sperm cell as carrying X-chromosomes orY-chromosomes. At least one QCL laser source emits at a wavelengthcorresponding to the resonant absorption for a target analyte and atleast one QCL laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Thearchitecture may further include a sort facility for sorting the spermcell according to the identified chromosomes. The architecture mayfurther include a second light source configured to deliver light to asperm cell within the sperm cells in order to induce a scatteringsignature, wherein the scattering signature is used to identify a spermcell characteristic, wherein the characteristic comprises one or more ofsize, cell type, cell density, and cell orientation. The second lightsource may be one or more of a VIS, UV, and NIR laser source. Gating theQCL-based vibrational measurements may be based on the second lightsource measurement. The architecture may further include a celldestruction or immobilization facility, such as a laser emitting at 1.5microns, that selectively terminates or immobilizes sperm cells based onthe chromosomes they are carrying.

In an embodiment, a cytometry method may include flowing cells past atleast one quantum cascade laser (QCL) source using a fluid handlingsystem, wherein the fluid handling system comprises a facility to enablea single cell flow in a measurement volume, delivering QCL light to thesingle cell in the measurement volume in order to induce resonantmid-infrared absorption by one or more analytes of the cell, anddetecting, using a mid-infrared detection facility, the transmittedmid-infrared wavelength light, wherein the transmitted mid-infraredwavelength light is used to identify a cell characteristic.

In an embodiment, a label- and stain-free cytometry system forpre-fertilization gender selection may include a handling system thatpresents a single unlabeled and unstained sperm cell to at least onelaser source, the at least one laser source configured to deliver lightto the sperm cell in order to induce a resonant absorption by DNA withinthe sperm cell at one or mid-IR wavelengths. The wavelength of the lightdelivered by the laser source is greater than one third of the diameterof the sperm cell. A detection facility may detect the transmittedmid-IR wavelength light, wherein the transmitted mid-IR wavelength lightis used to identify the sperm cell as carrying X-chromosomes orY-chromosomes. The system may further include a sort facility forsorting the sperm cell according to the identified chromosomes. At leastone laser source emits at a wavelength corresponding to the resonantabsorption for a target analyte and at least one laser source emits at awavelength used to cancel out a background signal from other analytesand from system artifacts. The system may further include a second lightsource configured to deliver light to the sperm cell in order to inducea scattering signature, wherein the scattering signature is used toidentify a sperm cell characteristic, wherein the characteristiccomprises one or more of size, cell type, cell density, and cellorientation. The second light source may be one or more of a VIS, UV,and NIR laser source. The system may further include a cell destructionor immobilization facility, such as a laser emitting at 1.5 microns,that selectively terminates or immobilizes sperm cells based on thechromosomes they are carrying.

In embodiments, cytometry systems for pre-fertilization gender selectionmay use substantially orientation-independent spectroscopy.

In an embodiment, a high yield cytometry system for pre-fertilizationgender selection using mid-IR spectroscopy may include a handling systemthat presents a single sperm cell to at least one QCL laser source, theat least one QCL laser source configured to deliver light to the spermcell in order to induce resonant absorption by the sperm cell DNA at oneor more mid-IR wavelengths. A detection facility may detect thetransmitted mid-infrared wavelength light, wherein the transmittedmid-infrared wavelength light is used to identify the sperm cell ascarrying X-chromosomes or Y-chromosomes. A sort facility for sorting thesperm cell according to the identified chromosomes may achieve puritieson the order of at least 75%, greater than 90%, or at least 99%. Forexample, the purity of Y-chromosome carrying sperm cells may be at least75%. In another example, the purity of X-chromosome carrying sperm cellsmay be at least 90%. At least one QCL laser source emits at a wavelengthcorresponding to the resonant absorption for a target analyte and atleast one QCL laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Asecond light source may be configured to deliver light to the sperm cellin order to induce a scattering signature, wherein the scatteringsignature is used to identify a sperm cell characteristic, wherein thecharacteristic comprises one or more of size, cell type, cell density,and cell orientation. The second light source may be one or more of aVIS, UV, and NIR laser source. Gating the QCL-based vibrationalmeasurements may be based on the second light source measurement. Thesystem may further include a cell destruction facility, such as a laseremitting at 1.5 microns, that selectively terminates sperm cells basedon the chromosomes they are carrying.

In an embodiment, a low energy cytometry system for pre-fertilizationgender selection with diminished risk of cell damage may include ahandling system that presents a single sperm cell to at least one lasersource, the at least one laser source configured to deliver photons withan energy of less than 1 eV to the sperm cell in order to induce a bondvibration in DNA of the sperm cell. A detection facility may detect thetransmitted photon energy, wherein the transmitted photon energy is usedto identify the sperm cell as carrying X-chromosomes or Y-chromosomes.The system may further include a sort facility for sorting the spermcell according to the identified chromosomes. At least one laser sourceemits at a wavelength corresponding to the resonant absorption for DNAand at least one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts.

Cytometry systems also described herein enable single cell study andinspection. Such systems may include light sources that induce resonantabsorption in the mid-IR wavelength region, such as a QCL laser.However, these systems may also include other light sources and othertechnologies, such as fluorescence-activated spectroscopy systems andmicrofluidic architectures.

in an embodiment, a minimally invasive cytometry system with QCLinspection of single cells for cancer detection may include a handlingsystem that presents a single cell to at least one QCL laser source, theat least one QCL laser source configured to deliver light to the cell inorder to induce vibrational bond absorption in one or more analyteswithin the cell and a detection facility that detects the mid-infraredwavelength light transmitted by the cell and identifies the cell aseither cancerous or non-cancerous. The system may further include a sortfacility for sorting the cell according to its status. At least one QCLlaser source emits at a wavelength corresponding to the resonantabsorption for a target analyte and at least one QCL laser source emitsat a wavelength used to cancel out a background signal from otheranalytes and from system artifacts. The system may further include asecond light source configured to deliver light to the cell in order toinduce a scattering signature, wherein the scattering signature is usedto identify a cell characteristic, wherein the characteristic comprisesone or more of size, cell type, cell density, and cell orientation. Thesecond light source may be one or more of a VIS, UV, and NIR lasersource and measurement with the second light source may be used ingating the QCL-based vibrational measurements. The system may furtherinclude a cell destruction or immobilization facility, such as a laseremitting at 1.5 microns, that selectively terminates or immobilizescells based on the identification.

In an embodiment, a cytometry system with a QCL laser source, acousticdetection facility, and micro-fluidic cell handling system may beconfigured for inspection of individual cells. The cytometry system mayinclude a micro-fluidic cell handling system that enables a flow ofcells past at least one quantum cascade laser (QCL) source, wherein thehandling system comprises a facility to enable a single cell flow in ameasurement volume of the microfluidic architecture, the at least oneQCL laser source configured to deliver light to a single cell in themeasurement volume in order to induce resonant mid-IR vibrationalabsorption by one or more analytes, leading to local heating thatresults in thermal expansion and an associated shockwave. An acousticdetection facility detects the shockwave emitted by the single cell. Themagnitude of the shockwave is indicative of a cell characteristic. Thecharacteristic may be a quantity of a nucleic acid, a protein, a lipid,a nutrient, and a metabolic product. The micro-fluidic cell handlingsystem further comprises a filter that excludes cells based on at leastone of a shape, a size, and a membrane integrity. The cytometry systemmay further include a sort facility for sorting the single cellaccording to the identified characteristic. At least one QCL lasersource emits at a wavelength corresponding to the resonant absorptionfor a target analyte and at least one QCL laser source emits at awavelength used to cancel out a background signal from other analytesand from system artifacts.

In an embodiment, a minimally invasive inspection system may use mid-IRvibrational spectroscopy for high throughput, high accuracy cytometry.The system may include a handling system that enables high throughputpresentation of single live cells to at least one light source, the atleast one light source configured to deliver light to the live cell inorder to induce vibrational bond absorption in one or more analyteswithin the cell at one or more mid-IR wavelengths. A mid-infrareddetection facility may detect the transmitted mid-infrared wavelengthlight, wherein the transmitted mid-infrared wavelength light is used todetermine a cell characteristic comprising one or more of chemicalcomposition, size, shape, and density. The throughput of live cells maybe at least 1 cell per second, at least 10 cells/sec, at least 100cells/sec, at least 1,000 cells/sec, at least 4,000 cells/sec or atleast 10,000 cells/sec. In this embodiment and in other embodimentsdescribed herein, at least one light source emits at a wavelengthcorresponding to the resonant absorption for a target analyte and atleast one light source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. In thisembodiment and in other embodiments described herein, the system mayfurther include a second light source configured to deliver light to thecell in order to induce a scattering signature, wherein the scatteringsignature is used to identify a cell characteristic, wherein thecharacteristic comprises one or more of size, cell type, cell density,and cell orientation. The second light source may be one or more of aVIS, UV, and NIR laser source. The wavelength of the light delivered bythe light source may be greater than one third of the diameter of thecell. In this embodiment and in other embodiments described herein, thesystem may further include a cell destruction facility, such as a laseremitting at 1.5 microns, that selectively terminates cells based on thecharacteristic.

In an embodiment, a low energy cytometry system with diminished risk ofcell damage may include a handling system that presents a cell to atleast one laser source, the at least one laser source configured todeliver photons with an energy of less than 1 eV to the cell in order toinduce a bond vibration in DNA of the cell. A detection facility maydetect the transmitted photon energy, wherein the transmitted photonenergy is used to identify a DNA characteristic of the cell.

In an embodiment, a minimally invasive cytometry system using QCLvibrational spectroscopy for differentiation of pluripotent stem cellsfrom functionally differentiated cells based on inspection of singlecells may include a handling system that presents a single cell to atleast one QCL laser source, the at least one QCL laser source configuredto deliver light to the single cell in order to induce vibrational bondabsorption in one or more analytes within the cell. A detection facilitymay detect the transmitted mid-infrared wavelength light, wherein thetransmitted mid-infrared wavelength light is used to identify thedifferentiation status of the cell as either pluripotent or functionallydifferentiated. The system may further include a sort facility forsorting the cell according to its differentiation status. At least oneQCL laser source may emit at a wavelength corresponding to the resonantabsorption for a target analyte and at least one QCL laser source emitsat a wavelength used to cancel out a background signal from otheranalytes and from system artifacts. The system may further include asecond light source configured to deliver light to the cell in order toinduce a scattering signature, wherein the scattering signature is usedto identify a cell characteristic, wherein the characteristic comprisesone or more of size, cell type, cell density, and cell orientation. Thesecond light source may be one or more of a VIS, UV, and NIR lasersource. The system may further include gating the QCL-based vibrationalmeasurements based on the second light source measurement. The systemmay further include a cell destruction or immobilization facility, suchas a laser emitting at 1.5 microns, that selectively terminates orimmobilizes cells based on the differentiation status.

In an embodiment, a cytometry system with a QCL source, mid-infrareddetector, and micro-fluidic cell handling system configured forinspection of individual cells may include a fluid handling system thatenables a flow of cells past at least one quantum cascade laser (QCL)source, wherein the fluid handling system comprises a facility to enablea single cell flow in a measurement volume. The at least one QCL lasersource may be configured to deliver light to the single cell in themeasurement volume in order to induce resonant mid-infrared absorptionby one or more analytes of the cell. A mid-infrared detection facilitymay detect the transmitted mid-infrared wavelength light, wherein thetransmitted mid-infrared wavelength light is used to identify a cellcharacteristic. The characteristic may be a quantity of at least one ofa nucleic acid, a protein, a lipid, a metabolic product, a dissolvedgas, and a nutrient. The fluid handling system may comprise amicrofluidic architecture. At least one QCL laser source emits at awavelength corresponding to the resonant absorption for a target analyteand at least one QCL laser source emits at a wavelength used to cancelout a background signal from other analytes and from system artifacts.The system may further include a sort facility for sorting the singlecell according to the identified characteristic. The fluid handlingsystem may further include a filter that excludes cells based on atleast one of a shape, a size, and membrane integrity. The system furtherincludes a cell destruction or immobilization facility, such as a laseremitting at 1.5 microns, that selectively terminates or immobilizescells based on the identified characteristic. A second light source maybe configured to deliver light to the cell in order to induce ascattering signature, wherein the scattering signature is used toidentify a cell characteristic, wherein the characteristic comprises oneor more of size, cell type, cell density, and cell orientation. Thesecond light source may be one or more of a VIS, UV, and NIR lasersource. The system may further include gating the QCL-based vibrationalmeasurements based on the second light source measurement.

In an embodiment, a mid-IR spectroscopy cytometry system with selectivecell destruction capability may include a handling system that presentsa live cell to at least one laser source, the at least one laser sourceconfigured to deliver light to the live cell in order to induce resonantmid-infrared absorption by at least one analyte within the cell. Amid-infrared detection facility may detect the transmitted mid-infraredwavelength light, wherein the transmitted mid-infrared wavelength lightis used to determine a cell characteristic. A cell destruction facilitymay selectively terminate cells based on the characteristic. The cellcharacteristic may include one or more of a nucleic acid quantity, anucleic acid type, a chemical composition, a size, a shape, and adensity. The cell destruction facility may be a laser emitting at 1.5microns. At least one laser source emits at a wavelength correspondingto the resonant absorption for a target analyte and at least one lasersource emits at a wavelength used to cancel out a background signal fromother analytes and from system artifacts. The system may further includea second light source configured to deliver light to the live cell inorder to induce a scattering signature, wherein the scattering signatureis used to identify a live cell characteristic, wherein thecharacteristic comprises one or more of size, cell type, cell density,and cell orientation. The second light source may be one or more of aVIS, UV, and NIR laser source. The wavelength of the light delivered bythe laser source may be greater than one third of the diameter of thesperm cell.

In an embodiment, a mid-IR spectroscopy system may include lasertweezers for moving cells into position for measurement with a lightsource that induces resonant absorption in an analyte within the cell.

In an embodiment, a mid-IR spectroscopy system with fluidic featuresthat pre-filter cells to obtain cells of appropriate size may include afluid handling system that enables a flow of cells past at least onelaser source, wherein the fluid handling system comprises a filter thatexcludes cells from a measurement volume of the fluid handling systembased on size and/or shape, the at least one laser source configured todeliver light to a single cell in the measurement volume in order toinduce resonant absorption in at least one analyte within the cell. Amid-infrared detection facility may detect the transmitted mid-infraredwavelength light. At least one laser source emits at a wavelengthcorresponding to the resonant absorption for a target analyte and atleast one laser source emits at a wavelength used to cancel out abackground signal from other analytes and from system artifacts. Thesystem may further include a second light source configured to deliverlight to the cell in order to induce a scattering signature, wherein thescattering signature is used to identify a cell characteristic, whereinthe characteristic comprises one or more of size, cell type, celldensity, and cell orientation. The second light source may be one ormore of a VIS, UV, and NIR laser source. The wavelength of the lightdelivered by the laser source may be greater than one third of thediameter of the sperm cell. The system may further include a celldestruction or immobilization facility, such as a laser emitting at 1.5microns, that selectively terminates or immobilizes cells based on thetransmitted mid-infrared wavelength light.

In an embodiment, a cellular DNA measurement system may include ahandling system that presents a single cell to at least one lasersource, the at least one laser source configured to deliver light to thecell in a measurement volume of the handling system in order to induceresonant absorption in the DNA within the cell. A mid-infrared detectionfacility may detect transmitted mid-infrared wavelength light, whereinthe transmitted mid-infrared wavelength light is used to calculate thecellular DNA content. The DNA content may be used to identify cell cyclestatus in a plurality of cells and the cell cycle status for a pluralityof cells is used to determine a growth rate of the cells. The DNAcontent may be used to determine aneuploidy, wherein the aneuploidycharacteristic is identified by a low DNA count, an extra chromosome,and/or a missing chromosome. The system may further include a sortfacility for sorting the cells according to the aneuploidycharacteristic. The handling system may further include a filter thatexcludes cells based on a characteristic. At least one laser sourceemits at a wavelength corresponding to the resonant absorption for atarget analyte and at least one laser source emits at a wavelength usedto cancel out a background signal from other analytes and from systemartifacts.

In an embodiment, a cellular DNA measurement system may include ahandling system that presents a fluorescently labeled single cell to oneor more of a visible or UV laser source and a handling system thatpresents the fluorescently labeled single cell to at least one QCL lasersource. The at least one QCL laser source may be configured to deliverlight to the cell in order to induce resonant absorption in the DNAwithin the cell. A mid-infrared detection facility may detecttransmitted mid-infrared wavelength light, wherein the transmittedmid-infrared wavelength light is used to calculate cellular DNA content.A visible light detection facility may detect the fluorescing label.

Mid-IR based systems described herein enable single particle study andinspection. Such systems may include light sources that induce resonantabsorption in the mid-IR wavelength region, such as a QCL laser.However, these systems may also include other light sources and othertechnologies, such as fluorescence-activated spectroscopy systems,microfluidic architectures, additional optics, scattering analysis, andthe like.

In an embodiment, a single particle QCL-based mid-IR spectroscopy systemwith differential numerical aperture optics for emitted and scatteredlight may include a handling system that presents a single particle toat least one quantum cascade laser (QCL) source, the at least one QCLlaser source configured to deliver light to the single particle in orderto induce resonant mid-infrared absorption in one of the particle or atleast one analyte within the particle. The system may also include anoptic to capture mid-IR wavelength light transmitted through the cell,wherein the optic has a smaller numerical aperture than a focusing opticfocusing the QCL laser emission on the cell. A mid-infrared detectionfacility may detect the transmitted mid-IR wavelength light andscattered mid-IR wavelength light. The particle may be a cell. Thehandling system may further include a filter that excludes particlesbased on at least one of a shape, a size, and a membrane integrity. Thesystem may further include a sort facility for sorting the singleparticle according to one of the transmitted light and scattered light.At least one QCL laser source emits at a wavelength corresponding to theresonant absorption for a target analyte and at least one QCL lasersource emits at a wavelength used to cancel out a background signal fromother analytes and from system artifacts.

In an embodiment, a single particle QCL-based mid-IR spectroscopy systemusing resonant scattering for measurement may include a handling systemthat presents a single particle to at least one quantum cascade laser(QCL) source, the at least one QCL laser source configured to deliverlight to the single particle in order to induce resonant opticalscattering based on wavelength-specific refractive index shiftsresulting from resonant bond vibration of one or more target analytes. Amid-infrared detection facility may detect the mid-infrared wavelengthlight scattered by the single particle. The particle may be a cell. Thehandling system may further include a filter that excludes particlesbased on at least one of a shape, a size, and a membrane integrity. Thesystem may further include a sort facility for sorting the singleparticle according to the scattered light. At least one QCL laser sourceemits at a wavelength corresponding to the resonant absorption for atarget analyte and at least one QCL laser source emits at a wavelengthused to cancel out a background signal from other analytes and fromsystem artifacts. The mid-infrared detection facility may include aplurality of mid-infrared detectors deployed at multiple angles thatdetect the mid-infrared wavelength light scattered by the singleparticle.

In an embodiment, a single particle QCL-based mid-IR spectroscopy systemwith in-droplet microfluidic system may include a handling system thatsuspends particles in or as a droplet within another liquid, wherein thehandling system presents individual droplets to the at least one quantumcascade laser (QCL) source, the at least one QCL laser source configuredto deliver light to a single droplet in the measurement volume of themicrofluidic system in order to induce resonant mid-infrared absorptionin at least one analyte within the droplet. A mid-infrared detectionfacility may detect the mid-infrared wavelength light transmitted by thedroplet. The droplet and the another liquid may be immiscible. Theparticle may be a cell. When the particle is a cell, the mid-infraredwavelength light transmitted may be used to measure byproducts of cellmetabolism in the fluid surrounding the cell. The particle may undergo achemical reaction with the surrounding fluid in the droplet and themid-infrared wavelength light transmitted may be used to measure thelevel of reactants or the products of this reaction. At least one QCLlaser source emits at a wavelength corresponding to the resonantabsorption for a target analyte and at least one QCL laser source emitsat a wavelength used to cancel out a background signal from otheranalytes and from system artifacts.

In an embodiment, a single particle QCL-based mid-IR spectroscopy systemwith analysis of scattering includes a handling system that presents asingle particle tagged with a mid-IR active tag to at least one quantumcascade laser (QCL) source, the at least one QCL laser source configuredto deliver light to the single particle in order to induce resonantmid-infrared absorption in the particle or an analyte within theparticle. A mid-infrared detection facility may detect the mid-infraredwavelength light scattered by the single particle. A wavelength andangle analysis of the scattered mid-IR wavelength light may be used todetermine analyte-specific structural and concentration information. Theparticle may be a cell. The mid-IR active tag may be a quantum dot. Thehandling system may further include a filter that excludes particlesbased on at least one of a shape, a size, and a membrane integrity. Thesystem may further include a sort facility for sorting the singleparticle according to one or more of a transmitted mid-IR wavelengthlight and the scattered mid-IR wavelength light. At least one QCL lasersource emits at a wavelength corresponding to the resonant absorptionfor a target analyte and at least one QCL laser source emits at awavelength used to cancel out a background signal from other analytesand from system artifacts. In embodiments, mid-IR active tags may beused in a direct transmission measurement as well, such as with any ofthe embodiments described herein.

In an embodiment, a handling system may present a single particlelabeled with a mid-IR active label to at least one quantum cascade laser(QCL) source, the at least one QCL laser source configured to deliverlight to the single particle in order to induce resonant mid-infraredabsorption by the mid-IR active label of the particle. A mid-infrareddetection facility may detect the transmitted mid-infrared wavelengthlight.

In embodiments of the present invention, the wavelength for inspectionmay include wavelengths in the mid-IR band, wavelength specific to peakscorresponding to DNA vibrational modes, and the like. Wavelengthselection may be optimized such as to inhibit scattering, and the like.

In embodiments, optical architecture and system components for thepresent invention may include facilities associated with reducingoptoelectronic noise, confirming and/or modifying cell angle or positionin a measurement volume, controlling cell rate spacing, accounting fornuclear volume in analysis, and reducing and accounting for QCL supplynoise. Laser sources may include tunable QCLs, multiple tunable lasers,broadband lasers, scanning lasers, THz QCLs, QCL-on-a-chip,Vernier-tuned QCLs, single pulsed QCL, multiple pulsed QCLs, parametricoscillators, and the like. The architecture may also enable measurementsfrom various angles of capture and using beams from multiple angles. Aphase scrambling device may be used to get rid of coherent artifacts. Asdescribed herein, differential aperture optics for input and output maybe used to focus the emitted light and then capture transmitted light ina wider area.

Systems with QCL sources may have a facility for separating wavelengthselectronically based on timing of pulses and relaying them to separatedetection facilities. Filters, such as dichroic filters and gratings,may be used to optically filter wavelengths. A prism or beam splittermay be used to separate wavelengths. The output from multiple sourcesmay be combined in one detector. An array of lasers may cross thechannel at different points—these can be combined and directed to asingle detector.

Reference detection facilities may also be located at an angle outsidethe main angle of capture to detect scattered light. Use of resonantscattering may be made to obtain shape and position information.Scattered light may serve a gating signal or calibration for the primarymeasurement, such as the QCL-based measurement. Use of mid-IR activetags and reagents with QCL interrogation may enable an analysis ofscattering (e.g. for angle, wavelength). The architecture may enablepolarization-based measurements of particles in flow.

In embodiments, form factors for the present invention may include acassette, a chip, a 2D manifold, 2D array, microfluidic architecture,flow cuvette, flow cytometer, cell-on-tape/substrate and the like.Handling systems may include laser tweezers, micro-fluidic handlingsystem (e.g. for circulating cells, micro-fluidic flow systemcomponents, charge applied to cell for selection), fluid flowcomponents, with tracers, with additives, with anti-reflective coating,with labels (e.g. quantum dot labels), pre-sorting particles based onsize, a scanner system where the source moves relative to the detecteditem rather than moving the item, a microfluidic manifold (cells moverelative to each other) and the like. The scanner system may includeformats such as Cell on tape, Cell on substrate, Cells on a 2D carrierthat is read out by translating at least one of the (i) carrier or (ii)the source and the detector. Flow architecture may be a species of amicrofluidic manifold. Multiple channels of flow and multiple detectionpoints may be present in a single system

Vibrational spectroscopy may include direct, indirect, Raman, coherent,anti-Stokes Raman, and the like. Functional benefits may include aminimally invasive system, with no labels, with no dyes, using no UVlaser, with increased accuracy, increased throughput (e.g. orientationindependent), low coefficient of variation, and the like. Two systemsmay be put end-to-end to sort for one thing in the a first system, thentake the output and do a task, such as a measurement or a second sort.

In embodiments, the present invention may provide facilities associatedwith scattering, such as measuring scattering, mitigating the effects ofscattering, correcting for scattering, utilizing resonant Miescattering, and the like. The present invention may provide for celldestruction through the use of a laser.

In embodiments, the present invention may be applied to a variety ofapplications, including gender selection (e.g. sperm DNA assessment,such as with humans, horses, cattle, pets), motility detection (e.g.sorting for motility, integrated onto the chip), cancer detection, stemcell study and manipulation (e.g. for monitoring differentiation ofpluripotent cells into functionally differentiated cells, in harvestingstem cells, in the purification of stem cells before transfer topatients), aneuploidy detection, in the measuring of cell growth rate ina sample, measuring RNA characteristics, measuring sugarscharacteristics, measuring protein characteristics, as a DNA statisticstool, in the measurement of other particles in liquids, in reactionmonitoring, in measuring circulating tumor cells, for embryo scoring, indetermining blood count, in semen analysis, in gas monitoring, insolid-in-liquid measurement, in blood diagnostics (e.g. for malaria,parasites), in food and water contamination analysis (e.g. measuring theIR footprint for E Coli, in emulsions (e.g. using a in-dropletmicro-fluidic system), and the like.

This disclosure provides a cytometry platform for measuringcharacteristics of particles in a flow using a mid-IR based measurementsystem and vibrational spectroscopy. The mid-IR based measurement systemmay be based on a quantum cascade laser (QCL) source delivering mid-IRwavelengths in the range of 3 to 15 microns. The QCL laser has a narrowwavelength emission comprising an easily tailored center wavelength anda low entendue, enabling measurement of a small area and narrow angleand measurements on the order of microseconds. For example, the power ofthe QCL may be ≤10 mW and a conduction band offset of about 0.1 eV. QCLvariations include multiple tunable lasers, broadband, and scanning.

The cytometry platform may include a microfluidic architecture. Thecytometry platform enables direct measurement of the chemical content ofthe cell using vibrational spectroscopy. Two types of vibrationalspectroscopy may be used: (a) direct absorption where the particle isexposed to a wavelength in the mid-IR fingerprint region of about 5-12microns, which corresponds to the frequency of a bond vibration and (b)Raman spectroscopy which is an indirect way of making measurements.Coherent anti-stokes Raman spectroscopy (CARS) may also be used tomeasure vibrational bond fingerprints in cells.

The cytometry platform enables a stain- and label-free process that issafe to cells and is orientation independent, high throughput, highaccuracy, and high yield. The advantages of the cytometry platforminclude the ability to directly measure an absorption line withoutlabels or dyes/stains. Another advantage is use of light that is 20-25times less energetic light than UV light that is used in conventionalsystems and thus much less likely to damage cell. Because of thewavelength being relatively long (six times longer than UV laser), thereis less scattering and the scattering is much better behaved compared toother light sources, such as visible light. Another advantage is that asopposed to traditional FTIR that requires cells to be dried, thecytometry platform can measure cells in a liquid medium where refractiveindex differential is much lower, so scattering index is lower.

In some instances of the cytometry platform, an additional scatteringmeasurement may be made specifically to see how big and dense a cell is.For example, a blood count can be done in this fashion. With mid-IRlight and in particular light whose angle can be controlled well (with avery collimated beam), chemical composition and size information may beobtained by measuring scattering off of particles.

In embodiments of the cytometry platform, a conventionalfluorescence-activated sorting (FACS) system may be used in parallel inorder to obtain a simple binary measurement for a cell then get anaccurate numerical measurement for chemical content using the mid-IRbased measurement system.

The cytometry platform enables measurement of cellular content, such asprotein bonds and nucleic acid bonds. For example, three characteristicDNA peaks, an asymmetric PO₂ ⁻ stretch (DNA) at 1236 cm⁻¹, a symmetricPO₂ ⁻ stretch (DNA) at 1087 cm⁻¹, and a C—C deoxyribose stretch (DNA) at968 cm⁻¹.

The cytometry platform is engineered to reduce the QCL supply noise, theRIN noise associated with emission of the QCL, shot noise associatedwith the transmitted mid-IR energy, detector noise, and pre-amp noise.For example, at bandwidths up to 10,000 cells/second, the optoelectronicsystem noise is 3.7 ppm. Other elements of the cytometry platform werealso designed to reduce system noise, such as the selection of thechannel height, the input angle, the collection angle, the flow rate,and cell spacing.

One design of the system includes 2 or more fixed wavelength QCLs whereat least one is at a “signal” wavelength (corresponding to the resonantabsorption for a target analyte) and at least one is at a “reference”wavelength (a nearby wavelength used to cancel out background from otheranalytes, and from system artifacts). The QCLs may be in a coolinghousing and may be driven by a pulsed driver or some other kind ofdriver. A grating-based QCL tuning system may be included to tune theQCLs. Before reaching the sample, the QCLs may first traverse a pelliclebeamsplitter and adjustable aperture. Transmitted mid-IR energy isdetected by a signal detector and a reference detector. A detector maymeasure scattering and compensate for it in the main measurements. Forsuch a system, the predicted collection rate is >4,000/sec and thepredicted purity is >99%. The QCLs may have carrier frequencies so thata single detector may be used and the signal is separated by modulationfrequencies.

With wide variations in nuclear volume and cell orientation, there isvariability in the absorption peak. Selecting a wavelength that is attop of the curve where the strongest absorption exists may not be idealas strong absorption may make measurements more variable with cellorientation.

The cytometry platform is designed to reduce cell nucleus heating.Heating, due to DNA absorption of mid-IR energy, may be reduced and mayhave a narrow distribution even with wide nuclear volume/orientationdistribution. For example, heating may be kept to under 1K.

In the cytometry platform, a QCL laser source may be configured to emitenergy to a single cell in a measurement volume of the platform in orderto induce resonant mid-IR vibrational absorption by one or moreanalytes, leading to local heating that results in thermal expansion andan associated shockwave. The cytometry platform may include an acousticdetector that detects the shockwave transmitted by the single cell,wherein the shockwave is indicative of a cell characteristic.

In the cytometry platform, a cell destruction facility may be includedto selectively terminate cells based on a characteristic. In the exampleof pre-fertilization gender selection, those sperm not exhibiting thedesired chromosomal type may be targeted for motility cessation using alaser emitting at 1.5 microns which may be used to immobilize cellswithout damaging DNA contents. In other cases complete destruction ofcells through membrane destruction or other means may be effected,through the use of a laser or other means.

The cytometry platform may take many forms. The cytometry platform maybe embodied in a mid-IR cuvette for a standard flow cytometer where cellselection is based on an applied charge. The cytometry platform may beembodied in a 2D manifold/array for immobilizing a plurality of cellsand then measuring them individually using the present invention,potentially repeatedly. The cytometry platform may be embodied in asystem with laser tweezers that traps cells with a visible laser andmoves the cell into position for measurement. The cytometry platform maybe embodied in a microfluidic chip with a waveguide to capture IR lightthat crosses the channel. The microfluidic chip may include fluidicfeatures that pre-filter cells by size. The cytometry platform may beembodied in a circulating cell culture system.

The height of the channel may be optimized so that a resonant opticalfield is not obtained even if the AR coatings are not robust.

The mid-IR wavelength used in various embodiments herein may beoptimized for low scattering.

The mid-IR based inspection and measurement system may further include afacility for a polarization-based measurement of particles in a flow,such as of DNA especially. Even with measurement of phosphate bonds, ifleft hand or right hand polarized light is emitted, because the DNA isin a helix, an improved separation of a DNA-specific signal may beobtained by using circularly polarized light.

Now that we have described particular embodiments of the presentdisclosure, we turn to describing a set of figures that will illustratethese and other embodiments.

FIG. 1 illustrates the present invention configured in a flowarchitecture 100. The flow architecture 100 may be a minimally invasivecytometry system, a microfluidic architecture, a cell inspection system,a label free cytometry system, a dye free cytometry system, a high yieldcytometry system, a low energy cytometry system, an aneuploidymeasurement system, a growth rate measurement system, an in-dropletmicrofluidic system, and the like. The flow architecture 100 comprises alaser source 102, a detector 104, a pre-filter 108, a single cell flow110, a sort facility 112, one or more sort destinations 114, and ahandling system 118. The laser source 102 may be a QCL, a QCL array, aQCL array of multiple angles, multiple QCLs with distinct carrierfrequencies, a Vernier tuned QCL, a dual QCL/UV array, a QCL with phasescrambling facility, a mid-IR laser, a tunable QCL, a broadband QCL, ascanning QCL, a THz QCL, or any combination thereof. The detector 104,also known as a detection facility, may be one or more of a mid-IRdetector, visible/NIR scattered light detector, fluorescence detector,quantum dot label detector, detector with differential numericalaperture, reference detector for calibration, photo-acoustic detector,or a combination thereof, and may be selected in accordance with thelaser or light source. The handling system 118 may be a microfluidichandling system, a chip/cassette with or without anti-reflectivecoating, a 2D manifold/array, a laser tweezers, and the like. Thehandling system 118 may comprise a facility to enable single particle orcell presentation to the one or more laser sources 102. In anembodiment, handling system 118 may be a fluid handling system in amicrofluidic architecture. Various cytometric analyses,characterizations, measurements, diagnoses and identification may bepossible using the present disclosure such as pre-fertilization genderselection, cancer detection, reproductive cell/embryo viabilitydetection, high throughput live cell studies (optionally in combinationwith FACS), stem cell studies, selective cell destruction, measurementof aneuploidy, measurement of growth rate, measurement of DNA, RNA,proteins, sugars, lipids, nutrients, metabolic products, etc, gasmonitoring, determining food/water contamination, and the like. A samplefluid may be encased in a sheath fluid that may allow droplets to becharged. The fluid stream may be streamed out of a nozzle. The presentinvention uses flow architecture 100 of a path length such as 50 micronsor less to reduce water absorption of infrared signal. In an embodiment,the stream may be passed through an optical measurement zone, whereinfrared light emitted by laser source 102 passes through the streamincluding at least one first cell type 120 and at least one second celltype 122. The optical interrogation may occur either after the fluidexits the nozzle, as shown in FIG. 1, or when it is still within thenozzle. In the in-nozzle case, the nozzle may be made of at least oneinfrared-transparent material such as Germanium, very pure Silicon,chalcogenide glasses, Calcium Flouride, Zinc Selenide and the like. Thebeam from QCL 102 passes through the fluid stream and may be detected onthe opposite side with one or more mid-infrared detectors 104. When aliving cell is detected in the stream, the absorption of analytes withinthe living cell at one or more mid-IR wavelengths may be measured as thecell moves through the beam by studying the transmitted mid-IRwavelength light. The signal from detector 104 may be processed to yieldan estimate of certain biochemical constituents in the cell. Theabsolute or relative level of these constituents may be used to classifythe first cell type 120 and/or the second cell type 122.

According to methods well known in flow cytometry, the stream isactuated in a manner, such as with a piezoelectric actuator that maycause it to break quickly into discrete droplets. These droplets may begiven an electrical charge according to the cell classificationdetermined using the QCL system. In an embodiment, the system may applya single charge for desirable cells, and route all other droplets to awaste bin. However, in other embodiments, various systems may setmultiple levels of charge in order to allow sorting in to sortdestination 114. Once assigned a charge, the droplet may beattracted/repelled by charged plates in the system. The negativelycharged droplets may be attracted by the +Ve plate, and sorted into oneoutput container; the positively charged droplets may be attracted bythe −Ve plate. The droplets whose readings may be inconclusive (wherethe droplet contains no cell, or where the droplet contains more thanone cell) may be sorted into a waste container.

In an embodiment, the configuration in FIG. 1 may be applied topre-fertilization sperm cell sorting for gender, for example. Spermcells would be interrogated by QCL 102 that may be tuned to theabsorption of DNA, possibly QCL 102 tuned to other cellular matter, andpossibly a visible laser to measure scatter from the cell. The visiblelaser and cellular matter QCL wavelengths may be used to determine if asingle sperm cell was present, and possibly the orientation of the cell.The absorption of the wavelength tuned to QCL 102, and the associateddetector 104, may be integrated and processed together with the otherreadings to determine the total mass of DNA in the cell. The 2-5%differential in DNA mass between cells carrying the X- and Y-chromosomeswould be used to sort cells into X-bearing and Y-bearing samples.Inconclusive measurements, multi-cell droplets, and droplets withoutcells may be sent to a waste container. In other embodiments, the systemmay be further simplified for this application by having only “selectedcells” and “waste” outputs. In some embodiments, spectral measurementsof sperm cells may have been used to measure the extent of anychromosomal/DNA damage in a cell. Cells that may have unusual spectra intheir DNA fingerprint may therefore be discarded. Similarly, cellsshowing unusual ratios of other cellular matter compared to DNA mattermay indicate lack of viability or damage to the cell, and these may beused to reject the cell.

FIG. 2 shows a potential configuration of laser source 102 tointerrogate a sample stream, in either a flow cytometer 100configuration as shown in FIG. 1, or in another configuration wherecells are presented in a fluid channel such as in a microfluidic chipsystem. In this embodiment, a visible laser 202 may be used to detectthe arrival of a cell in the stream by its scattering signature. Thescattering signal may encode other information about the cell includingsize, features that may help indicate the cell type, or orientation.

The visible scattering signal may then be used to trigger QCL 204operation where one or more pulsed QCLs are used. The advantage ofpulsed QCLs running at a low duty cycle is that they may producesignificantly higher power for the short period in which the cell is inthe measurement location, allowing for a higher signal-to-noise ratio inthe measurement.

The visible laser 202 measurement may be configured such that it detectscells ahead of the QCL 204 volume for a number of reasons as it mayallow the QCL 204 to be turned on and stabilize in terms of power andwavelength before the spectral measurement commences, it may also bedesirable that the QCL-based measurement begins before the cell arrives,and coincidentally with the visible laser, in the measurement volume, inorder to have baseline infrared measurements before and after the cellis measured. However, in other embodiments, a separate detector may beused on the output of the QCL 204 to normalize out laser power. In otherconfigurations, the “visible” and mid-infrared measurement volumes maybe the same, and beams may be combined into a single beam. The visiblebeam may be enlarged along the flow axis to produce a signal that islonger than the QCL-based measurement. The visible measurement maymeasure scatter to one or more detectors 206, and may even be used toproduce an image or pseudo-image of the cell in order to measure size ororientation. Cell orientation may be critical information for the sortbased on QCLs 204. For example, sperm cells, which pack DNA very tightlyinto their body, may be asymmetric. Transmission measurement through thelong axis of the cell may result in a significantly different absorptionmeasurement than through the short axis. With the aid of one or morevisible beams and their scattered signals, cell orientation may bedetermined to process the QCL-based measurement more accurately.

FIG. 3 illustrates a simplified example of mid-infrared spectra for aflow such as those described in FIGS. 1 and 2. The example showsinfrared transmission of three constituent materials in the flow ofwater which may have many absorption features in the mid-infrared,cellular components other than DNA, and DNA.

In an embodiment, three QCL wavelengths may be used: one to measurewater absorption through the stream, one to measure cellular componentsother than DNA, and one to measure the DNA signal. The three absorptionspectra may be overlapping, in which case a 3-QCL approach is desirablein order to strip out the DNA signal. In an embodiment, the outputs ofthe 3 QCLs may be combined into a single beam that is sent through thesample, and then broken into separate wavelengths using thin filmfilters and the like, and detected by 3 mid-infrared detectors such ascooled Mercury-Cadmium-Telluride (MCT) detectors. The absorption of thewater is calculated first, to normalize the measurement of the non-DNAcellular constituents; in the case where the cellular constituentabsorption overlaps with the DNA absorption spectrum, this signal isthen used to normalize the signal received from the DNA-specificwavelength detector. In an embodiment, the signal corresponding tonon-DNA components may be used separately to classify the cell type,orientation, and the like. In other embodiments, a broadband QCL sourcemay be used to produce mid-infrared light covering all the relevantfeatures, and a similar 3-detector configuration may be used. Anotherconfiguration may use a scanning tunable QCL that rapidly scans thewavelength range of interest, in addition to a single detector. Anotherconfiguration may be the use of a broadband QCL source plus a tunabledetector system.

FIG. 4 shows an example configuration of a system interrogating cells ina flow, which is shown in cross-section. In this embodiment, multipleQCL-originated infrared wavelengths may be combined with a visiblewavelength to interrogate cells. The wavelengths may be combined, andthen separated, using dichroic thin film filters. The QCL wavelengthsmay be used in this case to normalize for water, or to measure relativeconstituents within the cell. The visible wavelength may be used todetect the cell, and in other ways described earlier.

FIG. 5 shows an embodiment of the present invention, in which the flowand cells 120 and 122 are measured from multiple angles. Again, thissystem may use one or more QCL wavelengths and visible/near infraredwavelength(s). Measurements using one or more paths through the flow maybe used to calculate or normalize for cell orientation or preciseposition within the fluid flow. Such multi-angle measurements may resultin significantly higher precision measurements of cellular components.In other embodiments, visible beams where laser sources and detectorsare significantly less expensive may be used from multiple angles toprecisely localize the cell in the flow and/or measure its orientationto normalize measurements made using the QCL source(s) and associatedmid-infrared detector(s). The embodiment shown in FIG. 5 may be extendedto multiple angles through the sample. If additional power is needed toprovide high enough (signal-to-noise) SNR, multiple QCL sources may beused. As described above, this may be supplemented with visible/nearinfrared measurements from multiple orientations, which may use the samebeam paths, or a separate set of beam paths.

FIG. 6 illustrates a simplified detector signal, corresponding to thesample spectra shown in FIG. 3. A visible detector measures scatter froma cell entering the measurement volume, with the dip in signalindicating the presence of a cell. For example, one QCL is used tomeasure water absorption through the stream (essentially measuring thepath length through the stream). Another QCL is used to measure generalcellular components. Yet another QCL is tuned specifically to theabsorption band for DNA (for example the O—P—O stretch band at 1095 cm̂−1DNA/RNA). In the manner described above, the signals are normalized toisolate the signal of interest (which may be the DNA content of thecell).

FIG. 7 shows another embodiment of the present disclosure, where cellsare measured in a dry state. In this embodiment, a continuous tape isused as a substrate for the cells for measurement and separation. Thetape may be metal-coated to provide high reflectivity in themid-infrared. This example may be shown for sperm cells, which maywithstand some level of desiccation. Sperm cells may be spread onto thetape from a liquid reservoir—a “squeegee” type nozzle may be used,potentially with features that may provide the sperm cells with a commonorientation on the measurement tape. The sample may be air-dried rapidlybut done carefully, so that it does not destroy the cells to a pointwhere very little extraneous fluid is left around the cells. A sensorhead containing QCL lasers, detectors, and visible/near infraredlasers/detectors may be then scanned over the tape. Absorption of theQCL wavelengths may be measured to determine, in a manner describedabove, the absolute amount of DNA contained in the cells and potentiallyother information regarding the cells.

In an embodiment, cells or regions that may be rejected are thencovered, with an ink jet type device capable of a high output rate, witha substance that fixes the rejected cells to the substrate. Optionally,the tape may be rehydrated to preserve the selected cells. The tape thenruns into an output reservoir, where the free (selected) cells may beextracted into a liquid. The tape with the rejected cells fixed to itgoes into a waste container. The advantage of this configuration may bethat the resulting piece of equipment may be very small, and takeadvantage of components already developed for low-cost, high-speedscanner and printer systems. As the cost of QCL components may bereduced, this makes possible the use of low-cost, compact systems forclinics or even home use.

In addition, the removal of liquids from the “sample stream” greatlyincreases the transmission of mid-infrared signal and potentiallyimproves the SNR of the system. The basic system illustrated in FIG. 7may be configured in alternate manners in line with the presentinvention. Other embodiments may include use of freezing rather thandrying to fix the cells in place and prepare for measurement; use ofselective unfreezing and removal in order to accomplish the selectionprocess; and no use of drying or freezing but allowing a thin layer ofliquid or gel to remain on the surface of the tape by hardening of theliquid/gel by any means in order to fix certain cells to the tape whileothers are extracted, including selective drying, exposure to radiationthat hardens a gel, and the like. Another embodiment may include use ofa laser or other mechanism to destroy or disable cells that may bedetermined by use of the present invention to be of a type not desiredby the sort. Potential subsequent filtering to separate dead from livecells may also be included.

FIG. 8 shows the application of the present invention embodied using amicrofluidic-type cell sorting system. Such devices have beendemonstrated for cell sorting using conventional dye plus UV-activatedfluorescence classification of cells. In an embodiment, the microfluidicdevice is configured in a basic manner, with an input well and twooutput wells. Sorting at the junction is accomplished in one of theseveral ways known to those in the field, including but not limited toelectrical fields, lasers, magnetic means (in conjunction with magneticbeads), or fluidic pressure ports. In an embodiment, the system may beconfigured to allow interrogation using one or more QCLs from the top,and simultaneous interrogation using a visible laser from the bottom.This may be achieved, for example, by using a glass substrate as thelower half of the device (transmissive in the visible) and high-puritysilicon as the upper “lid” for the device (transmissive in themid-infrared). The lids may be coated to improve optical performance;for example, the glass in the channel may be coated with a (visiblelight) transparent conductive layer that strongly reflects mid-infraredradiation to maximize the mid-IR signal returned to the mid-IRdetector(s). As described above, the visible laser may be used to detecta cell in the detection volume, and trigger pulsed QCL operation. Themid-infrared light from one or more QCLs passes through the cell, isreflected by the bottom of the microfluidic channel, and into themid-infrared detectors. The integrated signal as the cell passes throughthe detection volume may be used to classify the cell type, and tocontrol the sorting mechanism at the junction.

FIG. 9 shows a portion of a very basic embodiment of the presentinvention which is a microfluidic system for live cell measurements. Amicrofluidic chip with input 900 and output 902 wells may beconstructed, in manners well known in the market, here with threelayers: a top cap 912 in which the input 900 and output 902 wells areetched, a bottom cap 910, and a patterned layer 914 in whichmicrofluidic features are patterned. This layer may consist of any of anumber of materials, for example Polydimethylsiloxane (PDMS), which isreadily patternable (by imprinting, for example) and biocompatible. Awell may be formed in this material corresponding to those patterned inthe top cover. One or more channels 904, which may be tapered to preventclogging, may be patterned to connect the input 900 and output 902wells. A portion of the channel constitutes the measurement region 908where a mid-infrared beam produced by one or more quantum cascade lasers(QCLs) may be focused on the channel 904 and transmitted or reflected toone or more mid-infrared detector(s), such as a mercury cadmiumtelluride (MCT) photodetector. Cells passing through or positioned inthe measurement area 908 may cause absorption of specific wavelengths ofmid-infrared light corresponding to molecular vibration modes. Theabsorption measured by the system at these wavelengths may be used tochemically and therefore biologically characterize the cells.

One or both of the caps 910 and 912 and may be a material that transmitsthe mid-infrared wavelength(s) of interest. Standard glasses used inmicrofluidics may absorb these wavelengths. To build this architecturefor use with QCLs in the molecular “fingerprint region” (2-20 micronswavelength, where molecules have their fundamental vibration modes), aninfrared-transmissive material, for example ZnSe, may be used. Othermaterials which may be used may include at least one of Ge, Si, BaF₂,ZnS, CaF₂, and KCl. Certain materials such as BaF₂ or ZnSe may betransparent in at least portions of the visible light range, which maybe advantageous for systems where visible light guidance, observation,measurements or manipulation may be desired. The visible light mayinclude short wavelength light such as ultra-violet, visible, NIR ofupto 2 microns. In certain embodiments of the present invention, 1.5microns light may be used to ensure compatibility with theinfrared-transmissive materials.

If the system may be built for transmission measurements through thechannel 904 in the measurement area 908, then both top and bottom caps910 and 912 must be IR-transparent. An alternative configuration may bereflective, where the mid-IR light from the QCL(s) passes through onecap, passes through the measurement volume, reflects off the oppositecap, passes through the measurement volume a second time, and then exitsthe cap and is collected by a mid-IR detector. In this case, only mid-IRentry/exit cap may be required to transmit mid-IR light. For exampleSilicon may be used depending on the precise wavelengths being measured.High-purity Silicon such as float-zone Silicon may be desirable toreduce absorption losses in certain ranges. The opposite cap may use astandard microfluidic material, such as glass. In this architecture, itmay be desirable to coat the glass with a mid-IR reflective layer atleast in the measurement region 908. For example, even a thin layer ofmetal may be highly reflective in the mid-IR. Alternatively, aconductive oxide such as indium tin oxide (ITO) may be used in order toreflect mid-IR but be transparent in the visible range, in order toallow visible or near-infrared (NIR) access to the measurement volumefrom the opposite side. The flow of cells through the channel 904 andmeasurement area 908 may be controlled in a number of manners well knownin microfluidic systems, including pressure differentials between wellsand, or an electrical potential over these wells. The system may beconfigured to provide a continuous throughput of cells through themeasurement area 908 for applications such as cell type counting orpopulation statistics, or to allow precise positioning and stationarymeasurements of cells in this area for high-resolution spectral andother inspection of cells for R&D applications. The structureillustrated may be replicated or multiplexed using multiple fluidicchannels, which may be interrogated in parallel or sequentially byQCL-generated mid-IR light. Parallel channels may allow for highersystem throughput, and/or redundancy in case of clogging.

In an embodiment, the present invention may be useful to integrate amethod for disabling or destroying cells in the system. For example, alaser with sufficient power to destroy key portions of the cell may beused to disable the cell. The disabled cell then flows into the output,where it may be separated using filtering or other means, or left in thesample if it does not disrupt the function of the live cells. An exampleof such an application may be characterization and selection of spermcells. Those cells which meet the selection criteria (for gender, forinstance) may be allowed to flow through the channel unchanged, thosethat do not may be irradiated with a pulse of visible or infrared lightwhich damages their cell membrane or propulsion mechanisms.Subsequently, a “swim-up” filtering which selects for motile sperm maybe used to extract the live sperm cells. Alternatively, the entiresample may be used and only the live cells are able to fertilize theegg. A system based on the present disclosure would flow a suspension ofsperm cells through one or more microfluidic channels. In themeasurement volume of a channel, one or more QCL beams would be used todetermine the volume of DNA present in the sperm cell, and determinewhether the cell is carrying X or Y chromosomes (a 2.5-3% difference inDNA volume may be seen), and optionally whether there are mid-infraredspectral indicators for other problems. The mid-IR measurement may betriggered and/or supplemented by a low-power visible/NIR beam which maymeasure cell scattering or size, based on the X/Y characterization inthe mid-IR and other markers in the mid-IR and/or visible/NIR. Thosesperm which are desirable may flow through to the output without furtherintervention. Those that are not may be illuminated within themeasurement area, or immediately after it with a pulse of light whichimmobilizes it. In an embodiment, the present invention may utilizelight in the near or short-wave infrared range to immobilize sperm. Inan embodiment, a mechanism for separating motile from non-motile spermmay be built directly into the microfluidic device in this system.

Another example where such a selective-kill mechanism may be employed isin stem cell therapies. For example, when a suspension of differentiatedcells grown from pluripotent stem cells may be prepared for deliveryinto the subject, it is important to remove or disable residual stemcells, which may grow into tumors within the target organ. In this case,a filtering system based on the present invention would inspect a flowof cells in one or more microfluidic channels, and interrogate thesecells with mid-IR beams from one or more QCLs. Based on the observedspectral characteristics, and the calculated biochemical makeup of thecells, certain cells that may be identified as stem cells or cells thatare not the type of differentiated tissue required, may be destroyedusing a laser pulse. In this manner, differentiated cells receiveminimal handling and only very low-energy mid-IR radiation exposure,while cells which could cause abnormal growths if delivered intact tothe target tissue are rendered non-functional.

A similar system may be used if a closed-circuit culturing, growth andfiltering system may be built for stem cell or other applications. Cellsmay be continuously run through a filtering system that inspectscellular biochemical fingerprints and terminates cells that do not meetthe application requirements. Again, applications of such systems mayinclude regenerative medicine based on stem cells. Another potentialapplication of such a system may be in high-throughput “evolutionary”processes where specific cell function is being targeted, and thisfunction or its byproducts may be observed using mid-IR spectroscopictechniques. For example, synthesis of specific chemicals may be thefunctional target, in this case a system based on the present inventionmay flow, with high throughput, cells through microfluidic channelswhere they are interrogated using QCL(s) and mid-IR detectors. Cellsshowing promise may be recirculated into the system without change,those that do not may be destroyed using a laser or other potentialtools such as ultrasound, RF, mechanical punches, liquid jets and thelike, so they do not contribute to future populations in the system.

FIG. 10a shows detail of an embodiment of a measurement region in amicrofluidic channel 1002. Cells 1004 flow through the channel 1002 andpass a region illuminated by a mid-IR beam 1008 originating from one ormore QCLs. As the cell passes through the beam, mid-IR light is absorbedin a spectrally-dependent manner according to the molecular constituentsof the cell. In many cases, the beam area may be larger than the cellitself, and the extracted signal will correspond to an average over thecell and surrounding areas. QCLs, as opposed to traditional mid-IRsources such as hot filaments, may be able to focus significant powerinto small areas, which provides a strong advantage for this system. Inmany cases it may be desirable to mask surrounding areas in order toreduce the signal background. In this case a masking layer 1012 may bepatterned on to one of the caps in order to create an aperture 1010through which mid-IR light may pass. This may maximize contrast as thecell passes through the measurement volume, and reduces contributionsfrom other materials such as the PDMS or other material used to createthe fluidic channels.

FIG. 10b shows the same example as 10 a in cross-section. Specificallyit shows how a mask is used to ensure only a subset of the incomingmid-IR light from the QCL(s) is transmitted to the mid-IR detectors. Inthis example, both top and bottom caps 912 and 910 must be made out of amid-IR transmissive material. If visual-range observation ormeasurements are also desired, at least one of these must betransmissive in both the mid-IR and visible ranges.

FIG. 10c shows a cross-section of an alternative embodiment that may usea reflective measurement for the mid-IR light 1008. In this example, themeasurement volume is illuminated with mid-IR light 1008 through thebottom cap 910 that is made of IR-transmissive material. A portion ofthis light passes through the measurement volume, where it may passthrough a cell 1002, and is then reflected by a patterned reflectivelayer. The reflective layer may be a metal layer, or a conductive oxideas described earlier in order to allow visible light access into thevolume from the top. Reflected mid-IR makes an additional pass throughany cells in the measurement volume, and then passes out through thebottom cap 910 where it may be captured by a mid-IR detector system. Themid-IR light 1008 that is not reflected passes into the top cap 912 andis absorbed. The advantage of this architecture may be the ability touse two different wafer materials for the top and bottom caps 912 and910. For example, the top cap 912 could be made of glass, which is lowcost, transparent for visible observation or measurements, and can bereadily patterned to form fluid cells/ports. The bottom cap 910 may bemade of IR-transmissive material but not necessarily require visibletransmission. For example, silicon may be used for certain wavelengthranges.

FIG. 11 shows an embodiment of the invention where the microfluidics andQCL-based spectral measurement system may be combined with a moreconventional fluorescence-based measurement system. A microfluidicdevice 1100 may include channels to hold live cells 1008 as illuminatedwith one or more QCLs, which may include one or more tunable QCLs 1102.The mid-IR radiation emitted by the QCL passes through the measurementvolume and any cells 1008. The transmitted light may be measured by oneor more mid-IR detectors 1104. This may be done in either transmission(as illustrated here) or reflection (as described earlier) modes. Herethe system may be shown to be complemented by a laser in the UV orvisible range 1108 which may emit light 1110 that is also directed atthe measurement volume, where it excites fluorescent probes or dyesattached to specific cellular features. The resulting fluorescence maybe measured using one or more bandpass filters 1114 and detectors 1118.Such a system configuration may allow correlation of conventionallabel-based techniques with mid-IR spectroscopic measurements. It mayalso enable combined-mode systems where labels may be used to identifyspecific cell types/features, and mid-IR spectral measurements usingQCL(s) supplement this information with spectral measurements, such aspossibly for biochemical variations not possible to measure with knownlabels/dyes. Note this system may be configured, as discussed earlier,as a two-sided reflective system where IR and visible systems arepositioned on opposite surfaces of the microfluidic system and operatein reflection.

FIG. 12 shows an exemplary embodiment of the present invention where themicrofluidic subsystem includes a cell-sorting fluidic switch 1208. Aninput channel 1202 delivers cells to the measurement volume 1204 whereit may pass through a QCL-derived mid-IR beam from one or more QCLs,with one or more wavelengths probing the cell. The microfluidics mayhave been configured, in manners known in the industry, to center thecells in the flow channel. Based on absorption at these mid-IRwavelengths, the cell may be classified into one of two categories. Insome embodiments, two pressure ports 1210 may be used to displace eachcell to one side of the microfluidic channel, so as to cause it to flowinto one of two output channels 1212. Such a system may be used toaccumulate one type of cell out of a general flow of cells, or toforward one or two populations for further inspection and/or processing.An example application of such a configuration may include genderselection, where sperm cells carrying X or Y DNA may be sorted intogroups and one type may be retained for fertilization. In an example ofa stem cell applicaiton, pluripotent stem cells may be separated fromdifferentiated cells during extraction, or before reintroduction to asubject. Other applications include refinement processes where cells arecultured, possibly mutated and repeatedly measured, with certain cellsre-introduced to the culture based on their chemical “fingerprint”. Forexample, those cells which produce and therefore contain a specificproduct may be selected for, on a cell-by-cell basis. The switchingfunction in the microfluidic channel may be performed using any of anumber of techniques known to the art, including electrostatic forces,acoustic forces, optical pressure/heating of a portion of the channel,containment of cells in bubbles that may be transported within anothermedium, and the like.

FIG. 13a shows an alternative microfluidic-based embodiment of thepresent invention, where a series of microwells 1304 may be integratedinto a microfluidic flow channel/chamber 1302. This structure may be1-dimensional along a single channel or 2-dimensional along an openplane. Multiple parallel channels may be used as well. These wells serveto trap individual live cells in well-defined spaces where they may bemeasured using mid-IR techniques. For example, a suspension of cells maybe flowed through the channel 1302, and stopped momentarily to dropcells into the wells 1304. The suspension and stoppage time must becoordinated such that the majority of wells contain a single cell.

FIG. 13b shows how the wells may be then scanned using mid-IR light fromone or more QCLs. Scanning may be accomplished by translating themicrofluidic chip, or the laser leading mechanism. As described herein,this may be accomplished in either reflective or transmissive mode. Thisscan may be repeated multiple times in the case where a time series ofmeasurements is being established to monitor changes in cells, withconditions such as temperature or chemical inputs through the channelpossibly being varied. Such time series may be used, for example, tomonitor cell differentiation (such as for stem cell applications), cellmetabolism for drug studies, and the like.

Using this design, cells may also be separated using optical orelectrical or combined techniques. For example, a visible or NIR beammay be used to address a well, and the pressure and heating from thisradiation displaces the cell resident in it back into the flow layer,where it may be flowed out of the cell. Electrical heaters in the wellmay do the same, or electrostatic forces may be used to separate thecell from the well. These operations may be performed in parallel. Forexample, a projector-type system may be used to project light intomultiple selected wells simultaneously, causing the cells containedwithin them to be pushed up into the flow region, and transported out ofthe device. This may open the potential for higher throughput processingusing large arrays.

FIG. 14 depicts another embodiment in which the microfluidic chamber maybe 2-dimensional. A 2-dimensional fluid chamber 1402 may be formedbetween the upper and lower caps. Optionally, if the area is very large,spacers 1404 may be inserted to maintain consistent spacing between thecaps. Cells flow into this area where they may be measured using one ormore QCL(s) 1102 and mid-IR detector(s) 1104. Measurements may beperformed either by translating the microfluidic chamber relative to thebeam 1008, or moving the beam itself from cell to cell. This may becontrolled by a human operator, or an image processing system may locatecells and steer the QCL-derived beam onto them for spectralinterrogation.

Alternatively, an “optical tweezer” beam where a laser spot 1408 (suchas visible or near infrared) or annular pattern may be used to trap andmove cells within the chamber 1402. This method, which is welldocumented elsewhere, may be used to immobilize moving cells, such assperm cells, or mobile microorganisms for interrogation by mid-IR. Itmay also be used to move cells to a specific measurement volume 1410. Inthis case, the mid-IR interrogation region may be stationary, and thecells may be translated in and out of the measurement volume using theoptical tweezers. The optical tweezers may be used to immobilize thecell temporarily, or at least confine it to the measurement volume forsperm cell inspection, for instance during the mid-IR measurement. Insome cases, such a visible or NIR beam may also be used to manipulatethe cell optically, by heating it, and puncturing its membrane, with theentire process observed by the mid-IR spectral system based on QCLs.

In an embodiment, the optical tweezer may be used to translate a cell ina scanning motion over the measurement region, in order to build up an“image” of the cell at a sub-cellular level. This method may be usedtogether with special optical features patterned on one of the caps,such as plasmonic or negative index structures that focus mid-IR lightto a subwavelength spot for high resolution interrogation of the cell.

FIG. 15 shows an alternate embodiment of the mid-IR optics combined witha microfluidic channel 1502 in a top view. In this embodiment, opticalwaveguides capable of carrying mid-IR light without high losses may beused to deliver light from one or more QCLs to the measurement volumewithin the microchannel. These waveguides may be fabricated from a rangeof mid-IR transmissive materials. For example, silicon may be bonded toa glass wafer and patterned into waveguides using standardphotolithographic techniques. In this example, an input waveguide 1508delivers the mid-IR light to the measurement volume containing a singlecell 1504, where it is absorbed according to its wavelength(s) and theconcentration of various chemical constituents in the measurementvolume, including the cell. The transmitted light may be collected bythe output waveguide 1510 and delivered to a mid-IR detector.Alternatively, a reflective design may be used to get two or more passesthrough the cell (with output to waveguide 1512)—more than onereflection may also be used.

The potential advantage of a waveguide-based system may be a veryconsistent alignment of the light source and output relative to themeasurement volume, and the beam shape (and therefore illumination)relative to the volume. Changes in beam location and profile relative tomeasurement volumes in the present invention may be calibratedinitially, before use, or periodically, such as through the use ofcalibrated particles, for example, tiny plastic spheres of knowndiameter flowed through the microfluidic measurement cell. In addition,the strong absorption of water and other liquids in the mid-IR range mayallow the system to be calibrated more easily using a liquid-onlyapproach.

FIG. 16 shows a system for the growth and purification of cells based onthe present disclosure. A unique aspect of the disclosure may be that itallows repeated interrogation of live cells without labels and UV lightor other methods, meaning the preparation steps (such as labeling) maynot be required, cell function may not altered, DNA may not be damagedsuch as that caused by some chemical stains and UV light, and noprocessing may be required to remove labels. For the aforementionedreason, this makes the disclosed approaches ideal for use in systemswhere cells or their descendants may be repeatedly interrogated for thepurpose of “guiding” growth or purifying a cell culture.

In an embodiment, an integrated bioreactor 1602 may form the center ofthe system, where cells may be incubated and may multiply, differentiate(such as for stem cell cultures), or produce biochemical compounds ofinterest. Cells may be introduced to the system through a cell input1604. On the way into the reactor, the cells may pass through themeasurement volume 1608 which may be interrogated by one or more QCLs,and optionally visible/NIR lasers. During input, the cells may undergopurification, time permitting, through the use of a cell sorting switch1610. Cells that do not meet certain criteria may be sent to the outputwell 1612 for disposal. Those cells that do meet the criteria may movethrough the reactor input 1614 into the bioreactor chamber. The reactorchamber may take multiple forms. For example, the bioreactor may eitherbe integrated into the microfluidic chip, or be implemented inmacroscopic form. Multiple mid-IR measurement/sorting microfluidicdevices may be attached to a single bioreactor in order to achievehigher throughput. The reactor may function in ways well known to thoseskilled in the art, with cell culture media/growth promoters, etc, andpotentially surfaces or structures that may allow temporary attachmentof cell structures. Media to feed cells and promote growth,differentiation or mutation or, to stress cells with particularcompounds may be introduced to the bioreactor through an input 1620, andwaste products may be removed through an output or exhaust 1622. Inaddition, temperature and other conditions in the chamber may becontrolled. In one embodiment, the control is through a feedback loopbased on system-integrated sensors.

After a specified interval or operations, cells may be extracted throughreactor output 1618 and may be flowed through the measurement volume1608 and cell sorting switch 1610. This process may eliminate cells notmeeting the criteria, such as by ejecting them into output 1612, andflowing the remainder back into the chamber. At the end of the cycle, afinal sort outputs 1612 the desired, purified cells into the output 1612for collection. This final output may be different from the one in whichthe ejected cells was flowed. An example application is in the use ofpluripotent stem cells to grow specific cells for implant into an organ.Initially, the input cells may be sorted to place only pluripotent stemcells into the bioreactor. These cells may then be cultured in order tomultiply, with continuous purification to remove any cells whichdifferentiate prematurely. Next, chemicals/media may be introduced intothe reactor and conditions changed that may promote differentiation intothe target tissue type. At this stage, the QCL-based sorter may serve tocontinuously remove cells that may have been differentiated into thewrong type of cell. Finally, at the output step, onlyproperly-differentiated cells may be sent to the output 1612, with anypluripotent cells removed to avoid potential tumor growths in the targetorgan.

Another example where such a system is of interest may be in guidingevolution of cells or even small multicellular organisms with specificchemical properties. For example, there may be used to evolve algae thatmay be efficient in producing precursors to biofuels. In this example, asystem based on the present disclosure, which may be used tocharacterize and measure intracellular chemical constituents with highthroughput, and without damaging the cells, may be of interest inperforming high-throughput “guided evolution” of optimized algae cells.In this case, the bioreactor serves to multiply successful cells, tointroduce a certain number of mutations, and the QCL-based single-cellchemical measurement system and sorter may serve to measure levels ofdesirable products being produced by live cells (without puncturingthem), and removing those cells which do not meet criteria. Such asystem has the potential to eliminate slow, tedious work withmacroscopic samples, and significantly accelerate development ofoptimized cell cultures. Similar methods may be used to “breed” cellcultures for use in other applications, including pharmaceuticals,“green” chemicals, nutritional supplements such as omega-3 fatty acids,and other substances that may be produced by microorganisms.

FIGS. 17a and 17b shows two potential configurations for QCLs and mid-IRdetectors in the present disclosure. In FIG. 17a , multiple QCLs 1702may be combined into a single beam by mirrors, such as half mirrors, orthin film interference filters 1704. The use of wavelength-specificfilters reduces the losses associated with combining multiple beams, butmay reduce the flexibility of the system if multiple tunable QCLs may beused with overlapping wavelength ranges. The beams may pass through themicrofluidic measurement volume 1708 and may be collected by a singlemid-IR detector 1710. In this configuration, the QCLs may be modulatedin a manner that allows their signals minus absorption in themeasurement volume to be easily separated by electronic means in thedetector output, by methods well known to those skilled in the art. Forexample, where pulsed QCLs may be used, they may be alternately pulsedsuch that they result in discrete measurements on the detector.Alternately, they may be modulated at characteristic (and potentiallydifferent frequencies), and the signals separated at the output throughthe use of analog or digital frequency filters.

FIG. 17b shows a configuration where the QCLs are arranged in the samemanner, but there may be separate detectors 1714 corresponding to eachwavelength, as filtered by filters 1712. The potential advantage of thisconfiguration may be higher system bandwidth, and the use of narrowpassbands in filters 1712 to further reduce background signals in themid-IR.

FIG. 18 shows an embodiment of the present invention where it may beused to sort live sperm cells for the purpose of pre-fertilization sexselection. A sample 1802 may be provided to the system through an input1804. A filtering mechanism 1808, which may for example be a densemedium through which only sperm cells may travel (possibly including acentrifuge or pressure function, and possibly selecting for motilesperm) is used to separate sperm cells from other constituents of thesemen. The filtering mechanism may optionally include a centrifuge orpressure function. The filtering mechanism may optionally select formotile sperm. This step may be done externally from the system throughmethods well known to those skilled in the art. Such a filter couldconsist of microfluidic features itself.

The filtered sperm cells possibly with the addition of a medium whichpromotes flow and viability may be transported to a sorter input chamber1810. From this chamber, they may flow into the measurement volume of amicrofluidic channel 1812, such as through microfluidic features whichorient cells and prevent clogging. Multiple microfluidic channels andmeasurement systems may be used where higher throughput is required.When a sperm cell arrives in the measurement volume 1814, it may bedetected through the use of a visible or NIR laser 1818 and a scatteringdetector 1820. The scattered signal may be used to determine whether thecell entering the measurement volume is indeed a sperm cell, and whetherit is oriented correctly, and/or visibly malformed. The signal may alsotrigger the use of QCLs 1822. In an embodiment, two QCLs may be used.One QCL may be used to measure the asymmetric phosphate bond vibrationat approximately 1087 cm⁻¹ which is characteristic of the DNA backbone.Another QCL may be used to establish a reference level, which may bedone with a wavelength just off the primary QCL, or at a known referencewavelength that establishes a reliable baseline for the measurement. Oneor both of the primary and reference QCLs may be rapidly scanned inwavelengths over a narrow range in order to facilitate measurement of asecond derivative, a common signal used in infrared spectroscopy. TheQCLs may be used in pulsed mode to achieve higher peak power, and toallow use of a single mid-IR detector 1828.

The reference wavelength may not be strongly absorbed by DNA or anyother target analyte. A subtraction or other analytical procedure may bedone, such as by a processor, of the reference signal in relationship tothe primary signal. Measurement of the signal itself may involvemeasuring an amplitude of absorption, standard deviation from the peakof the signal and/or reference curves, AUC/integration of the signal,and the like. In some embodiments, the reference signal may be just thesignal measured prior to an absorption curve.

A pre-filter may enable sorting cells by size, a surface property suchas an antigen or a cell surface protein, a chemical composition, acharacteristic determined by embodiments described herein, and the like.

Mid-IR light from the QCLs passes through the measurement volume 1814and any cells contained within it, and transmitted light may be measuredby mid-IR detector 1828. The calculated absorption in the DNA band, asnormalized by the reference QCL wavelength and measurements beforeand/or after the cell passes through the volume may be used to calculatetotal DNA contained within the sperm cell. This calculated concentrationmay be used to determine whether the cell is carrying X, Y or unknownchromosomes. Depending on the desired sex of the offspring, the cell isthen either let through unchanged, or disabled/destroyed through the useof an infrared laser 1830. To disable a sperm cell, the infrared sourcemay be pulsed at high power, focused on the cell or preferably, the tailof the sperm cell which may immobilize the sperm cell. The use of awavelength in the NIR (800 nm or higher) may minimize the possibility ofchromosomal damage, should a sperm cell treated in such a manner stillresult in fertilization. Multiple, lower-power pulses may be used.Subsequently, the sperm cells, both viable and immobilized, may betransported to the microfluidic output 1832, where they may be thenfiltered using a filter 1834. This filter should ideally select formotile sperm cells through a method such as the “swim-up” method, wheresperm swim through a thick cultured medium, thereby enabling collectionof only motile cells. As mentioned hereinabove, microfluidic, orwell-known macroscopic methods may be used to achieve this filtering.The output 1838 is then placed into a delivery mechanism 1840.

In various embodiments, the sperm cells may be cryogenically preservedbefore use. In this case it would be desirable to preserve themimmediately after sorting, and then run a motility-based filter afterunfreezing, since the freezing process itself may render a portion ofcells immobile. The present disclosure thereby enables a sperm selectionsystem that does not expose sperm cells to harmful dyes or to UVradiation, that may achieve high specificity in terms of genderselection, and that may be implemented in a closed system, whicheventually makes it viable for small clinic or even home or farm use.

FIG. 19a shows a cross-section of a fluid stream 1902 oriented to carrycells through a measurement volume such that flow is into or out of theplane of paper in this case. The flow is illuminated using one or moreQCLs, with a hypothetical illumination profile 1908 shown. Note thatthis may be an unlikely illumination profile and is shown to illustratethe invention only. Various beam shaping optics may be used to shape thebeam relative to the flow and to the region where cells are expected toflow through the detection volume. The mid-IR illumination is shown as1904, illustrated in this example to be wider than the liquid flow. Itshould be noted that a beam narrower than the flow may be used as well,and may be preferable from an optical power and contrast standpoint. Themid-IR light is strongly absorbed by water in the stream 1902 andtherefore, a characteristic absorption pattern 1910 is seen in thetransmitted radiation even in the absence of a cell in the measurementvolume. The light may be more strongly absorbed in the center since thestream in this case is circular. Ultimately, it will be highly desirableto measure the transmitted mid-IR radiation using a single detector;however, this only resolves the total power transmitted and not the beamprofile.

FIG. 19b shows the same configuration with two example cells in theflow. The positions may be exaggerated for illustration, and two cellsmay be shown simultaneously only to show positional variation over time.One cell 1912 may be well-centered in the flow, while another 1914 isoff-axis. The transmitted intensity profile 1918 shows how thispositional variation results in different power levels observed by thedetector. Each cell in this example may cause the same incrementalfractional absorption of IR light. For the on-center cell 1912, thefractional absorption 1920 may be of a smaller total amount of powerreaching the detector and therefore causes a smaller change in totalpower detected 1924. More mid-IR light arriving at the detector haspassed through the off-axis cell 1914 and therefore the fractionalabsorption 1922 may have a larger effect on total power change 1928.

As a result of this position-dependence, depending on the positionvariation of cells in the flow, and on the signal ultimately beingmeasured, it may be desirable to either minimize effect of position bydesign, or compensate effectively for cell position as it flows throughthe measurement volume.

FIG. 20a illustrates one configuration of the present invention thatminimizes the effect of cell position in the flow that results fromwater absorption. In this case, a rectangular or square flow 2002 may beused to force equal path lengths through liquid for all parts of themid-IR beam. A rectangular channel 2004 may be formed in material thatis transparent to mid-IR at least on the optical input and output sides.These surfaces may be antireflection (AR) coated for water to minimizestray reflections from surfaces and any resonant effects. The input beamprofile 2008 may be used to illuminate the channel. In this example, theportions of the beam outside of the sampling volume may be blocked,either by material choice or by making a mask that rejects this light(which would reduce contrast ratio in the measurements). As a result,the transmitted radiation 2010 may have a very consistent power profilespatially (limited by the input beam profile and diffraction effects).

FIG. 20b illustrates the rectangular channel with two hypothetical cellpositions in the measurement volume, a well-centered cell 2012 and anoff-axis cell 2014. In this configuration, the fractional absorptioncaused by the cells at different positions may be of the same amount oflight (same path length), as is illustrated by these absorptions 2020and 2022 on the output intensity profile 2018. As a result the powerchanges 2024 and 2028 that may be detected on the detector may be thesame for these positions.

A square or rectangular flow cell may be implemented in a number ofways. Such a nozzle could be drawn using standard glass-formingtechniques and made out of IR-transmitting glassy materials such aschalcogenides glasses. However, probably the most proven way to buildsuch a channel may be by building a 2- or 3-layer structure with onelayer patterned to form a channel. For example, two wafers ofIR-transmitting material could be AR-coated (for AR in water on oneside, and air on the other), a material that may be readily patterneddeposited on one of them, channels patterned into this material, andthen the wafers bonded together and diced to produce multiplemeasurement channels suitable for the present invention. Multiplemethods for forming such microfluidic channels are known in the industryand may be used to implement the present disclsoure. Of course, thematerials on optical input and output sides must be IR-transmissive. Forexample, Si, Ge, ZnSe and other materials with good mid-IR transmissionmay be used to form the channel that acts as the measurement volume.Optionally the material may also be chosen to transmit UV, visible ornear-infrared (NIR) light in order to make other measurements of theflow and cells including scattering, such as measurement of cellpresence, size, density and the like, and fluorescence, where acombination of traditional fluorescent label-based measurements andMid-IR measurements are desired.

FIG. 21 shows an embodiment of the present invention implemented in astandard flow cytometry system, in this case fitted for mid-IR cellmeasurements based on QCLs. The flow cytometric nozzle uses a sampleinjector 2102 to provide a steady flow of cells in a “core” flow, whichis combined with a sheath flow 2104 that surrounds it and merges with itin a laminar flow, which then narrows 2108 as it reaches the measurementvolume and ejection nozzle. An alternative to the core/sheatharchitecture that is most commonly used may be the use of acousticfocusing of the cells into the center of a single flow. In manytraditional flow cytometers, the stream is interrogated using UV andvisible lasers while it is a continuous flow in air 2110. In someembodiments, such lasers may also be used to measure cell properties,both through scattering and optionally through standard fluorescentlabels. Due to pressure waves applied to the nozzle liquid, the streamthen breaks into individual droplets 2112, which may be important forcases where cells are sorted into populations. In an embodiment, a cellsorter case, charge may be applied to the sheath fluid just before eachdroplet breaks off, based on the measurements that have been done on itsvolume and any cell it contains. However, in other embodiments, aplurality of cells could be used. Electrostatic fields are then used toguide these droplets into two or more output positions, where they maybe collected or discarded. Such systems are in wide use, andwell-understood by those familiar with the field of cell sorting.

In an embodiment, the present invention adds use of one or more QCLs2118 and mid-infrared detectors 2124 to the cell measurements, fordirect chemical measurements through vibrational spectroscopy. Ratherthan measure the flow in air, where it should be circular to facilitatesmooth flow and break into droplets, the mid-IR measurement may be donein an enclosed measurement volume using a mid-IR transparent flow cell2114. This flow cell is made of mid-IR transparent material andAR-coated in order to reduce reflection losses and artifacts (AR coatedfor water internally, and for air externally). The QCL-generated 2120light may originate from one or more QCLs, each of which may be tunableat either high or low speed. Low speed tuning would be used forcytometer setup for different tasks, whereas high-speed wavelength scanstuning, for example through the use of drive current, may be used toscan over a particular absorption peak of interest. The lighttransmitted 2122 through the flow measurement cell and any cellscontained within it may be then sent to one or more mid-IR detectors2124. Multiple detectors may be used to enhance the speed and/orsignal-to-noise ratio (SNR) of the system through the use of onedetector per wavelength range that is emitted by multiple QCLs. Forexample, a simple system would employ two QCLs, one for the signal ofinterest, and one for reference level. The light from the QCLs may becombined using a dichroic thin film filter or other wavelength-dependentcomponent, such as a grating, into a single input beam; the transmittedmid-IR light 2122 may be split using a thin film filter into two mid-IRdetectors, such as MCT detectors, one measuring the signal wavelengthand the other measuring the reference wavelength. An enhancement to thisbase system may rapidly scan one or both of these lasers of narrowranges, allowing a local derivative and second derivative in absorptionto be measured, which can greatly enhance accuracy against a varyingabsorption baseline.

The measurement cell 2114 may be fabricated with a square or rectangularcross-section to eliminate optical effects due to varying path lengths,as described hereinabove, as well as lensing and other optical effectsthat result from light impinging on surfaces non-perpendicularly. Boththe external air-facing and internal liquid-facing surfaces may beAR-coated to reduce light loss and stray reflections, and minimize anyinterference effects. In this architecture and in the otherarchitectures described herein, the present invention may be combinedwith traditional optical measurements using UV, visible or NIR lasers orother sources to interrogate cells in the flow. For example, fluorescentlabels attached to the cells may be read using UV and/or visible lasers.The combination of traditional fluorescent label readout and mid-IRvibrational measurements may open up new possibilities for research andclinical applications. For example, fluorescent antibody labels mayestablish a “yes/no” measurement for a particular type of cell, andmid-IR measurements may then be used to establish quantitativemeasurements of biochemical concentrations of materials such as DNA, RNAand the like. Such a combined-mode measurement system may further beused to research and develop mid-IR measurement techniques that offerhigher accuracy, lower cell damage and/or easier processes with betterapplicability to clinical or field devices than their fluorescent labelequivalents.

Another example of combined modes may be the use of scatteringmeasurements to determine cell size and shape. Screening cells by sizeand shape, such as to determine type of cell, may be combined withmid-IR measurements of chemical constituents to provide measurements ofa particular cell population, or to provide higher accuracy measurement.

On the output side of the cytometer, there may be various options otherthan breaking into droplets and sorting as shown in FIG. 21. The cellsand fluid may flow directly into a waste container, keeping the systemclosed, wherever measurement may be the objective. Where sorting may berequired, but a closed system is desirable, a number of alternativesorting techniques have been developed, including mechanical sorterswhere two or more channels are placed into the flow.

Another technique that may be combined with the present invention toproduce a closed, “all-optical” cell sorting system is photodamage cellselection. In this architecture, after measurement may have been doneusing mid-IR absorption measurements possibly in combination with moreconventional techniques, those cells that may not be desired in thepopulation are subjected to pulses of light, typically from a laser,sufficient in power to damage or destroy them. One example is the use ofpulses of light to immobilize sperm cells with laser pulses in the1000-2000 nm range and therefore prevent them from fertilizing an egg,without damaging DNA. In other cases, destruction of the cell membraneusing a laser pulse may be used to remove the cell from a population.Other non-optical techniques such as acoustic, mechanical, and RF havealso been described and may be used in embodiments of the presentdisclosure.

FIG. 22 shows the present disclosure embodied in an architecture thatallows cell position in a flow to be measured accurately, in order tocompensate for position-dependent variations in mid-IR absorptionsignal. The flow 2202 directed in or out of the plane of the pagecontains cells 2204 to be measured using mid-IR light 2210 from one ormore QCLs 2208, the transmitted portion of which may be measured by oneor more mid-IR detectors 2212. In some embodiments, the mid-IR beam iscombined with one or more visible/NIR beams 2218, from light source(s)2214 which may be used to accurately determine cell position and,potentially, size and shape. Dichroic filters 2220 and 2222 may be usedto combine and separate, respectively, the visible/NIR and mid-IR beams.When the visible/NIR light strikes the cell 2204, it may be partiallyscattered. This scattering is detected by visible/NIR detector(s) 2228.This configuration shows only forward scatter detectors, detectors maybe used at other angles to measure wider-angle scattering. If an arrayof light sources and/or detectors is used, they may be placed in amanner as to enhance the accuracy of the position measurement, forexample, they may be placed in a diagonal line across the flow in orderto add a time component to the measurement, as cells flow by the lightsources/detectors.

The ability to accurately determine cell position in this manner mayallow the mid-IR absorption signal to be compensated for cell position.In some embodiments, if the flow 2202 is circular in cross-section andthe cell 2204 is displaced from the center, the fact that more mid-IRlight signal is originating from the path containing the cell relativeto when the cell is centered in the flow may be used to compensate theapparent absorption signal. In addition, visible/NIR sensors may be usedto monitor the position and shape of the overall flow relative to themid-IR laser light path. The flow itself lenses the visible/NIR lightbased on its position, diameter, and shape.

In all of the embodiments in this invention, it may be useful to comparemid-IR transmission signals before and after cells pass through themeasurement volume with measurements during cell residence in thevolume. This may give a baseline that may show the water absorptioneffect that may be very strong in the mid-IR.

FIG. 23 shows an embodiment of the present invention that may useadditives to the core or sheath fluid in a flow to create a “tracer”which may be measured directly by the mid-IR subsystem, and thereforeallows accurate real-time calibration/compensation for core flowposition, and variations in flow shape and position.

FIG. 23a shows a sheath flow 2302 surrounding the core flow 2304 beingilluminated using mid-IR light 2308 from QCL(s). In this example, atracer may be added to the core flow at a known concentration. Thetracer may be selected to absorb at the wavelength of one of the QCLsources in the system. If the core flow diameter may be controlledaccurately, such as through accurate core/sheath pressure control, itsabsorption may then be used to track mid-IR absorption effects, such aswater absorption, on the core flow-related signal by using readingsbefore and after cells pass through the measurement volume.

FIG. 23b shows the same flow with a cell 2310 in the measurement volume.In an embodiment, a configuration of this system may employ at least 2QCLs, or 1 QCL that may rapidly tune over an absorption peak shared bythe tracer and the target cell component. The method to determineconcentration of the target compound in the cell may be then to measurethe absorption at the target mid-IR wavelength relative to waterabsorption without (FIG. 23a ) and with (FIG. 23b ) the cell present.This may give a signal relative to a known concentration, withflow-position-related factors removed.

FIG. 24 depicts another embodiment of the present invention in whichmultiple mid-IR beams may be used to interrogate the measurement volume.The use of multiple beams allows the position as well as the absorptionof a cell 2404 to be measured more accurately, akin to computedtomography methods used in medical imaging. One or more QCLs 2408 emitmid-IR light that may then split into three beams bypartially-reflecting mirrors 2412. These beams, with the help offully-reflective mirrors 2414 may be then directed at 3 different anglesthrough the flow 2402, which flows in or out of the plane of the page,and any cells contained within it 2404. The beams may then detect usingthree mid-IR detectors 2410. Measurements made before or after a cellmay pass through the measurement volume in this example may be used tonormalize the measurement, as they reflect the position and shape of thewater flow. Measurements of the cell made as it passes through thevolume may be from three angles. Using three measurements, plus themeasurements of the flow without the cell present, it may be possible todetermine both position and absorption of the cell within the flowaccording to algorithms well known in computed tomography.

In an embodiment, the system may be embodied as a microfluidic chip. Anexample of operation of the chip may include procuring a sample of cellsin suspension from patient or culture, with any necessary protocol toindividuate cells in the suspension; placing the entire sample into aninput port of a one-time-use plastic carrier; and placing the samplecarrier into a tool. Then, a cassette of microfluidic measurement/sortchips may be inserted into the tool. Multiple chips may be used for asingle sample in the case of clogging or degradation. Chips may be builtfrom materials that are optically compatible with the spectral cellmeasurement being performed. In the case of mid-infrared (QCL) basedmeasurement, the chip may be made of mid-IR transparent materialsincluding but not limited to Si, Ge, ZnSe, CaF₂, BaF₂ or salts withprotective coatings. Chips may have coatings that protect them fromfluid, are hydrophilic (to promote flow of liquid through measurementchannel) and provide anti-reflective (AR) functionality. AR coatings maybe applied to external surfaces to minimize reflections, and to the topand bottom of microfluidic channels in the measurement zone in order toprevent reflections, maximize signal, and minimize resonant opticaleffects (in this case the AR coatings are designed to minimizereflection at the interface with water). These internal (channelsurface) AR coatings are particularly important where a high-indexmaterial is used to construct the chip, in which case a resonant opticalfield could be created within the channel; this would lead to asituation where optical field intensity, and therefore interaction withbiochemical components of a cell being measured, would be dependent onthe vertical position of the cell within the channel, introducingvariability that may not be acceptable. This may be remedied withappropriate optical coatings that minimize reflections at theseinterfaces, and therefore minimize these resonance-created variations.Chips may be pre-loaded with liquid in the input and output volumes, aswell as the measurement channel, to eliminate issues with starting fluidflow through the measurement volume. Chips may include sealinglayers/tapes on the input and output reservoirs to maintain sterility,and prevent evaporation of priming liquid; the fluid in the input volumemay be pre-seeded with particles used for calibration of the chip. Chipsmay be charged with additional liquid which is used to create a “sheath”flow around the core flow containing the cells; this same fluid may beused to apply pressure to the system and maintain flow of cells throughthe measurement volume.

Known microfluidic configurations for creating 2D or 3D sheath/coreflows may be used. Sheath/pump fluid may be sealed in wells on the chipthat are opened for the purpose of pumping, or may reside below flexiblemembranes that can be addressed with an external pressure source,thereby maintaining fluid isolation.

The tool opens the input chamber of a chip, positions the chip onto thesample carrier, and moves a specific volume of sample onto the chipinput reservoir; the system may be configured to provide continuous flowfrom the sample carrier to the chip, or charge the chip in specificincrements, either one-time or in multiple batches. One potentialarchitecture opens a tape on input reservoir of the microfluidic chip,loads a small volume of the cell sample, re-seals the tape, and thenmoves the chip to the optical measurement system, where it remainssealed until the sample has been analyzed/sorted or until the chipclogs; then moves the chip back to the carrier, unseals the outputreservoir, and transfers the sorted sample to the output reservoir ofthe carrier; the chip is then disposed of (in the case where it is ameasurement only, the chip may be disposed of directly, without thetransfer to carrier step).

At startup on a specific sample, the tool runs a small but statisticallysignificant sample of cells through the measurement channel of the chip,and into the non-selected (disposed) output reservoir. Each cell ismeasured using the vibrational spectroscopy system described herein. Forexample, it is probed at three wavelengths corresponding to DNAabsorption, absorption of an interfering substance (which also absorbsat the DNA absorption wavelength; as an example certain proteins), and athird reference wavelength (to measure general absorption level due towater and other wide spectral features, for example). From these threemeasurements, a single DNA content number is calculated. The system alsomeasures average throughput rate of cells.

A histogram of cell counts vs. DNA content is generated and displayed tothe user. This gives the user an immediate sense for the distribution ofDNA content in cells, and whether there are distinguishable populations.It may give the user an immediate sense, for example, of whether apopulation of cells is multiplying (at this point some cells have twicethe normal amount of DNA). Optionally, the tool may “fit” Gaussian orother distribution curves to the observed population. These curves maybe application-specific (where a certain distribution of DNA content isassumed). Such curves serve to calculate expected purity in a sort ofthe cell population by DNA content.

The user may then select, through the user interface of the tool (whichmay be either on a built-in control panel, or on an attached devicessuch as a personal computer, laptop computer, tablet, smartphone, etc)at least two parameters of a sort, in the case where a sort is to beperformed: The range of DNA contents to select for in the “selected”output sample and the number of cells required in the output sample.

With respect to the range of DNA contents to select for in the“selected” output sample, this could correspond, depending on theapplication, to significant cell populations including but not limitedto: cells with X or Y chromosomes, where gender selection is beingperformed; cells with an abnormal number of chromosomes, where cancercells are being separated from a sample; cells that are in the processof dividing, where a cell study is being performed which involveslooking at viability or growth rates (the inverse—those that are notdividing, may also be selected for); DNA content-based sorting may alsobe used in the process of separating differentiated fromundifferentiated cells in stem cell related processes. If curves havebeen fitted to the populations, the tool will indicate the expectedpurity of the resulting sample as the user moves the selection “window.”It will also adjust the expected sort time for a desired number of cellsin the output.

With respect to the number of cells required in the output sample, theuser may adjust this number, up to a limit set by the expected number ofcells in the total sample, or by a maximum sort time.

The user then initiates the sort. In the case of pure measurement,rather than sort, the preceding steps are modified accordingly—thenumber of cells to be measured are selected; optionally the number ofcells sorted may be set by requiring a certain sample size within aspecific DNA content window, in order to obtain a statisticallysignificant sample size.

The tool then initiates the sort. Cells are flowed through themeasurement channel on the specialized optical chip, at a rate anddilution that allows sufficient measurement time in the opticalmeasurement volume, and allows them to be accurately sorted with theon-chip sorting mechanism. For example the following steps may beperformed by the system based on mid-Infrared quantum cascade lasersmeasuring mid-IR vibrational absorption features in the cells ofinterest.

Prior to a cell entering the measurement volume, the system continuouslymeasures the absorption of the (for example) three mid-IR wavelengthsbeing used; the signals are generated by three QCLs that are modulatedin a manner such that their signals may be separated after detection bya single detector, such as a thermoelectrically (TE)-cooled mercurycadmium telluride (MCT) photodetector. The signals observed at thedetector before (and after) the cell passes through the volume are usedas a baseline for the measurement, in order to cancel out, in largepart, slow-speed variations in the system, including variations inrelative laser power, changes in background absorption in the flowchannel, and slow-speed changes in optical effects in the system andchip (for example, mechanical changes or index of refraction changes dueto temperature). Modulations applied to these lasers may be of varioustypes. Pulsed modes may be used to generate short, distinct peaks, whereindividual wavelengths are pulsed in rapid succession. In continuouswave (CW) operation, the lasers may be modulated with specific carrierfrequencies that may then be separated using analog or digital filtersat the detector. Aside from power level modulation, the lasers may bemodulated in terms of wavelength. For example, wavelength may “chirp” aslasers are pulsed or modulated, because of current and temperatureeffects. This may effectively broaden the range of wavelengths at whichabsorption is measured. This “averaging” effect may be beneficial sinceabsorption features in liquid phase are typically quite broad. Externalwavelength-modulation components may be used within the QCLs to morebroadly sweep wavelength. For example, external cavity Fabry-Perotconfigurations may be used with wavelength-setting elements such asdiffraction gratings or tunable etalons. In one case, very rapid tuningover a broad range could enable measurement of both baseline (“valley”in the absorption spectrum) and signal (“peak” in spectrum) using asingle laser for a particular analyte. Center wavelength of the QCLs (orslave lasers, in the case of a CARS system), which may be set usingexternal tuning mechanisms or simply operating temperature (effected bya TE cooler on which the QCL is mounted) may be set to match theabsorption peaks (or valleys) observed in the cell sample. This processis performed during the setup of the system, and may be repeated atspecific intervals (during which all cells are sent to the “discard”output), or when the system senses recalibration is required. Thisprocess may be performed by sweeping the wavelength slowly as cells aremeasured, accumulating a distribution of signal points vs. wavelength,and then determining where the minimum or maximum signal occurred, andusing this as the wavelength set point.

As a cell enters the measurement volume, it is detected using avisible/NIR/SWIR beam that is scattered by the cellular material. Thisbeam is separate from the mid-IR beam and is used to detect cellpresence, flow speed (along with other sensors, possibly), and unusualscattering signatures that could signal cell agglomerates. The“presence” signal generated by this beam may also be used to triggerintegration of the mid-IR signals measured by the system. In the casewhere the system is built with mid-IR lasers, the material of the chipmust primarily be transparent to mid-IR wavelengths. Some materialsoptions such as silicon are transparent in much of the mid-IR, but nottransparent in the visible regime. In this case, a SWIR laser (1.5microns, for example) and detector combination—such as those developedfor telecom applications—may be used to measure this cell presence andscattering. Cell presence and scattering may be measured intransmission, reflection, or both.

Within the measurement volume, mid-IR light is transmitted through thecell, where it is absorbed according to its wavelengths and the chemicalconstituents (chemical bonds) within the cell. For example, where threewavelengths are used to make an accurate DNA measurement: There may be arise in the signal (i.e. lower absorption of) corresponding to thebackground, which is dominated by water, because other materials makingup the cell, such as proteins, lipids, and DNA displace water; whenadjusted for this change in background, a signal corresponding toproteins will show higher absorption; and when adjusted for the changein background, and after subtracting a known interference from protein(calculated by the previous measurement), the DNA absorption signaturerises.

The measurements may be integrated over the time that the cell resideswithin the measurement volume, and the integrated signals may be used toquantify cellular DNA. Other quantities (such as protein content, orcell size based on scattering) may also be measured, and may optionallybe presented to characterize the cell sample, or refine sort parameters.Combinations of DNA and protein content, DNA and lipid content, andother 2-axis or multi-axis analyses and sorts may be performed byappropriate extensions of the present invention, using appropriatemeasurement wavelengths for specific molecular bond vibrations.

Based on the measurement and the setpoint(s) for sorting (whenperforming a sort), a decision is made either to allow the cell toproceed to the “discard” output reservoir of the chip, or be diverted tothe “collection” output reservoir. The microfluidic channels may beconfigured so as to make the default route to the “discard” reservoir,and only when the switch is actuated will cells be able to reach the“collection” or “select” reservoir.

At a time interval after measurement, determined by the flow velocity inthe channel, a microfluidic switch may be actuated in order to push thecell into the appropriate output channel. At the position of thisswitch, or somewhere along the microfluidic channel preceding it, theremay be another measurement point illuminated by visible/NIR/SWIR lightwhere cell presence is measured by scattering. This allows realtimemeasurement of flow/cell velocity in the channel, and allows the systemto recalibrate timing of fluidic switch activation.

The cell, which may have had its position in the flow laterallyperturbed, is then routed into one of two or more output channels basedon the switch action. There may be additional optical measurement pointsin these output channels; these serve primarily that the switchactuation is functional and timing is correct, and observe any sortingerrors that occur. Should errors occur, the system may adjust timing ormagnitude of switch actuation, or simply discard the current chip anduse a new chip.

Cells are accumulated in at least two output reservoirs, the “discard”reservoir and the “select” reservoir.

The tool may detect one of a number of problem/failure conditions duringthe sort, including but not limited to: sample out, or front-endclogging, flow rate variation, switching failure and cell densityvariation.

With respect to sample out, or front-end clogging, no additional cellsappearing in the measurement channel may signal that the input sample onthe chip has been exhausted, or that the input of the channel isclogged. The system may: Perform anti-clogging procedures, which couldinclude rapid pressure pulses on the input or back flushing of thesystem and/or move to unload and discard the current chip, and load anew one to match up to the sample carrier.

With respect to flow rate variation, through the use of opticalmeasurement points, the system may detect that the sample flow rate isout of bounds. In this case, the system regulates the pressure in thesystem to adjust the speed of cells in the measurement channel. The flowrate may be estimated from a single sensor at the measurement point (bymeasuring how long the cell is present in the beam) or by 2 or moreoptical sensors along the measurement channel (by measuring time a celltakes to travel between points). If the cell speed is out ofbounds—which may cause DNA measurement SNR problems, or switchinginaccuracy problems, the system may send all cells to the default“discard” reservoir until the speed is properly regulated.

With respect to switching failure, through the use of opticalmeasurement points on the output channels, the system may detect thatcells are improperly switched to one of the output channels. In such acase, the system may be paused immediately (if a very high purity sortis being performed). Switch timing relative to measurement may beadjusted, as well as magnitude of signal being used on the cell switchmechanism.

With respect to cell density variation, the system may detect that cellsare arriving with spacing that is either too dense—not allowing cells tobe switched reliably—or too sparse prolonging the sort operation for agiven number of cells. The system may have provisions for adjusting thisdensity (such as dilution or concentration steps), or it may simplyupdate the user as to the change in anticipated purity or sort time.Dilution of cells may be performed during the transfer of sample fromthe sample carrier to the measurement chip; if this is the case,dilution may be adjusted from chip to chip until an optimum is found. Ifdilution is far off, a chip may be discarded, and another loaded ontothe sample carrier.

When the sample loaded into a particular chip has been sortedcompletely, the chip's “select” output reservoir is opened by thesystem, and its contents are transferred to the output reservoir of thesample carrier. This may be done with a liquid purge run through thechip or pipette action.

Once the chip's reservoir has been emptied, it is sealed, removed fromthe carrier, and discarded, potentially into a sealed compartmentattached to the disposable sample carrier.

This process continues until the sort is complete. At this point, theuser is signaled, and the sample carrier is prepared for unloading. Thesample carrier is removed from the tool, with the sorted sample readyfor use in the output reservoir. The sorted sample may subsequently beremoved by pipette or other method and used in subsequent lab orclinical procedures.

FIG. 25 shows two example configurations of the microfluidic chipsystem. FIG. 25a shows a tool that may accept microfluidic chips thatmay be pre-loaded by a user with the cell sample to be measured and/orsorted. This may be generally a configuration for relativelysmall-volume samples that may be contained on and sorted with a singlemicrofluidic chip. A slot 2502 may accept the chip into themeasurement/sorting system. A display 2504 shows histograms and otherindicators regarding cellular DNA content. The display may be atouchscreen display that may allows the user to make sort set points,set the graphical display of data, and start and stop operation. Thetool may include other interfaces, such as a USB interface to allowtransfer of data to (or control by) a computer, or transfer or backup ofdata onto a USB memory stick. Wireless networking interfaces may beincluded as well.

FIG. 25b shows another configuration of the tool described herein.Again, a display 2504 may allow the user to make setpoints and reviewdata from the cell measurements and/or sort. In this configuration,however, the cell sample may be loaded onto a larger carrier into thesample slot 2508. Separately, a cassette of measurement chips, one ormore of which may be used to sort/measure a single sample loaded in acarrier, may be inserted into chip cassette storage 2510. In thismanner, multiple chips may be used, if needed, to sort a single largersample loaded into the machine.

FIG. 26 shows example displays of DNA content shown to the user of thetool. FIG. 26a shows a configuration where DNA vs. cell count may bedisplayed in a histogram format. The x-axis 2602 in this example mayrepresent the DNA content of the cell. The y-axis 2604 represents thecumulative cell count in the sample. The distribution of cells measuredmay be indicated by 2608 (here showing a distribution representative ofcells that are actively dividing, and with some number of cells showingan abnormally low DNA count which could indicate aneuploidy indicated bythe small peak to the very left). This histogram in itself may bevaluable to the user for a rapid assessment of the cell sample. Inaddition, curve-fitting algorithms may be applied manually orautomatically, either in real time or offline after the measurement toestimate the percentage of cells in each state.

FIG. 26b shows a configuration where an additional parameter besides DNAmay be used to classify cells. In this case, the x-axis 2610 againrepresents the cellular DNA content. The y-axis 2612, however,represents another parameter measured by the system. Examples of thisparameter may include, but are not limited to, secondary vibrationalspectral measurement of the cell using the same technique but differentwavelengths to determine for example, protein content, lipid content,sugar content, RNA content. Examples of this parameter may also includevisible/NIR/SWIR light scattering from the cell, possibly indicatingsize and/or morphology. Examples of this parameter may include shape,size and density parameters calculated from imagery of the cell invisible/NIR/SWIR wavelengths. Examples of this parameter may includefluorescence signal from dyes or labels, such labels could include butare not limited to dye for assessing cell viability through membraneintegrity, membrane-staining dye to measure overall membrane, antibodiesattaching to specific cell types, and the like. Examples of thisparameter may include quantum dot and other labels which function in asimilar manner to fluorescent labels, though readout method isdifferent. Examples of this parameter may include multiple other cellmeasurement methods known to those in the field. It should be noted thatthis is not restricted to one additional parameter. Multi-dimensionalcell classification may be supported by the present disclsoure.

The density plot 2614 shows the cumulative density of cell counts in thesample. This may be represented by a simple scatter (dot) plot, andsupplemented with color or iso density lines to indicate statisticaldensity. In this example, there are two levels of DNA (for example adividing population of cells), and then two levels along the otherparameter. For example, if the other parameter here measured membranelipids as measured by a secondary vibrational signature, the plot mayindicate that there are two sizes of cell or cell agglomerates where twocells are adhered to one another while passing through the measurementvolume (and therefore should be rejected from the data, or sorted out ofthe sample).

FIG. 27 shows an example of a single-unit disposable measurement unit,consisting of a microfluidic chip 2704 with plastic carrier 2702 thatmay be used in a system such as the one shown in FIG. 27a . Themicrofluidic chip may be embedded in a plastic carrier, including inputport/reservoir 2708 and output port/reservoir 2710. For measurement-onlyapplications, the output port may be optional—the sample in this casewould be disposed with the carrier. Likewise, in sort applications, asingle output port (the configuration shown here) may be applicable ifthe rejected cells are disposed of together with the carrier. The inputport 2708 may be a well into which a cell sample is pipetted,alternatively it could take the form of a pipette head itself, so thatit may be used to draw a sample out of a well.

Where the optical measurement of the cells is made along themicrofluidic channel, a window 2712 may be formed in the plasticcarrier. This eliminates the effect of the plastic on opticaltransmission and provides a clear optical port. This is critical inparticular in the infrared, where plastic may have very lowtransmission. For example, the microfluidic chip may be manufactured ofSilicon or Germanium, which may be transmissive in portions of themid-infrared, and antireflection (AR) coated to minimize losses andfringe effects.

The carrier 2702 may be charged with the sample in the input port 2708,and then inserted into the tool. In an embodiment, tubes to controlpressure could then be matched to the input port 2708 and output port2710 in order to control the pressure differential and therefore thesample flow rate through the measurement channel. The plastic carriermay have features to provide rapid rough alignment within the system.Alignment of the chip with the optical readout system may further berefined by passive or active means. For example, the microfluidic chipmay have photolithographically-defined mechanical features which allowspassive alignment of the chip with the optical readout system, themicrofluidic chip may have photolithographically-defined features whichmay be optically interrogated in order to actively align the chip to theoptics or the optics to the chip, or the optical system may perform a“search” in which it uses the inherent absorption signals of themeasurement channel, the water in the channel, and any cells flowingthrough the channel in order to optimize focusing and x-y position ofthe beam relative to the channel.

After the measurement or sort is performed, the carrier may be ejectedfrom the tool. If this is a measurement-only process and tool, thecarrier may be rejected directly into a built-in disposal binappropriate for biohazardous samples. If the tool is being used for asort process, the carrier may be ejected or placed in an output bin forthe user to remove. The user may then remove the sorted sample from theoutput port/reservoir 2710 and proceed with the appropriate protocol.

FIG. 28 shows an alternative configuration, where a carrier 2802 may beused together with one or more microfluidic chips 2804, where the chipmay be a separate piece from the carrier. This enables the use ofmultiple microfluidic devices for a single larger sample, where cloggingor other chip problems are an issue, where precise calibration ofdilution is an issue, or where it may be desirable to run multiplemeasurements on multiple chips in parallel. In this case, multiple chipsmay be loaded with sample from the carrier, and run in parallel.

The carrier may have an input reservoir 2808 and zero or more outputreservoirs 2810. In this case where DNA measurements are done, there maybe no accessible output ports, and the sample is discarded with thecarrier. One or more output ports may be used in cases where a sort isperformed. This example shows a single-output carrier used formeasurement applications (the sample is available to the user aftermeasurement).

A microfluidic chip 2804 may be matched up to the carrier, as shown, anda portion of sample may be pushed from the input reservoir of thecarrier 2808 to the input port of the chip 2812. The carrier may havecompliant gaskets to allow efficient matching with the chip, and a goodseal around the transfer locations. In this example, the measurement isthen done in place (on top of the carrier). A pressure differential maybe applied directly to the carrier ports to transport liquid samplecontaining cells through the measurement channel on the chip. The samplemay flow through the measurement volume 2818 where it is interrogatedusing the vibrational spectroscopy system. A window 2820 in the plasticcarrier may be provided for clear optical access to the chipmicrofluidic channel. Following measurement, the sample may bepushed/pulled through the chip output port 2814 and into the carrieroutput reservoir 2810.

If clogging or other problems associated with the microfluidic chip maybe detected, the microfluidic chip may be removed and disposed of, and anew chip is matched to the carrier. This disposal may be done at aregular interval to preempt clogging or other issues, and/or toeffectively charge a “fee” per incremental sample measured or sorted. Inan embodiment, the carrier may include a disposal chamber for themicrofluidic chips, so that the system remains as closed as possible,and consumables may be disposed of after each sample run. Similarly, themicrofluidic chips may be included in the carrier itself, and removedfrom an internal magazine in the carrier as needed. In thisconfiguration, all consumables related to the process are delivered in asingle carrier or cartridge.

FIG. 29 shows some example formats for microfluidic chips that may beused in the present invention.

FIG. 29a shows a chip that could be used in a measurement-onlyapplication. An input port 2902 may be used to introduce the liquidsample with cells. A microfluidic channel 2910 transports this samplethrough the measurement volume 2908 to the output port 2904. Featuresmay be built into the chip to prevent clogging at the entrance to thechannel from the input port. Features in this region, and within thechannel itself, may also be used to orient the cells in a specificmanner, such as to prevent clogging, or to promote better measurement.For example, a pattern of posts of specific size and shape may be usedto break up agglomerates of cells, or to block large agglomerations ofcells or other substances from reaching the channel. Certainconfigurations of posts may in fact be used to pre-select cells of acertain size for measurement in the channel.

The chip may have photolithographically-defined mechanical or opticalfeatures which assist in aligning the chip to the optical interrogationsystem. For example, an etch process which may be alignedphotolithgraphically to the measurement channel (possibly the same etchprocess step as the one used to create the input and output ports), maybe used to create features for passive alignment of the chip to theoptical system. Alternatively, these may be optical features (reflectivemetal, or windows in a metal film) that allow the system to activelyalign the chip to the optics. Fine alignment may be done with featuresin the measurement volume itself, using the optical system itself tooptimally align for measurement.

FIG. 29b shows a similar chip, this one for use in sorting cells. Asample may be introduced into the input port 2914 and flows through amicrofluidic channel through the measurement volume 2922 andmicrofluidic cell 2924 switch and then into the output ports 2918 and2920. After measurement in the volume, DNA and other parameters for anindividual cell may be calculated. Based on these measurements, andsorting parameters entered into the system, the cell may be routedeither to the “discard” port or “select” port. Routing may be performedusing a microfluidic switch configuration well known to those working inmicrofluidics, two pressure ports 2928 may be used to shift the flow inthe channel to one side, causing cells to move into one branch oranother of the output junction, the mechanism is described in moredetail in FIG. 32 below. There may be an asymmetric configuration suchas the one shown in FIG. 29b where it is important to maintain purity inthe select output 2918. Thereby, in some embodiments, a “default” routeis to the discard output 2920, and only when an actuation is performedare cells routed to 2918.

FIG. 30 shows additional example configurations of microfluidic chipsfor use in the present invention.

FIG. 30a shows a configuration where a diluting/sheath fluid may be usedtogether with the sample in order to provide a centered flow in themeasurement channels. The sample may be introduced into input port 3002.A diluting fluid may be introduced, or pre-charged, in port/reservoir3004. This fluid may be used to apply pressure via duct on the inputport 3002 to drive sample through the measurement channel. In addition,side channels 3010 may be used to form a sheath fluid around thecore/sample flow at junction 3012. This centers the cells in the flow asthey pass through measurement volume 3014. The output port 3018 thenreceives the sample as well as the sheath fluid. This configurationpotentially provides a number of advantages, in that cell flow rate andspacing may be better controlled, and cells are centered within a widerchannel, where they can be measured with better repeatability. Inaddition, background signals from the sample fluid are reduced in thisconfiguration, as the core fluid flow may be typically very narrowcompared to the sheath flow.

FIG. 30b shows a configuration where cells may be switched, based on thevibrational spectroscopy measurement, using an electric field at theswitch point 3032. Cells flow from the input port 3024 through themeasurement volume 3028 where they may be measured as previouslydescribed. Based on individual cell characteristics, a voltage isapplied to contacts 3030 which perturb the path of the cell, and causesit to flow to a selected output reservoir 3034rec.

FIG. 31 shows an example construction of a microfluidic chip. In thisexample, a chip may be constructed for use in a mid-infraredspectroscopic cellular DNA measurement system utilizing quantum cascadelasers as the optical source(s). A top wafer 3114 and bottom wafer 3102may be used. These wafers may be made from materials that aretransmissive at the QCL wavelengths to be used for cellularinterrogation. For example, float-zone Silicon, which has hightransmission in the infrared, and which is readily obtained andmachined, may be used. Other materials that may be applicable includeGermanium, ZnSe, CaF2, BaF2, and other materials well known in infraredoptics.

The bottom wafer 3102 may be first antireflection (AR) coated. This isimportant for a number of reasons, such as to minimize losses of IRlight in the system and thereby maximize signal-to-noise ratio, orminimize QCL power required, reduce fringing artifacts from interfacereflections which may distort the transmission spectrum of the sampleand therefore distort apparent sample absorption and, importantly,minimize any resonant optical cavity effects in the channel 3112. If thechannel becomes a resonant optical cavity, the field intensity may varysignificantly with vertical position in the channel. In this case, thesystem may become sensitive to cellular position, because the cell maybe in a position to absorb more infrared radiation at field maxima, andless at minima. This may result in higher and lower apparent infraredabsorption based on cell position, which should be avoided.

An alternative or complementary solution may be to create a flow inwhich cells are confined to a specific layer, and therefore aresubjected to the same optical field from cell to cell. A good AR coatingmay be a more robust and simple solution, and design of mid-infrared ARcoatings may be well known to those skilled in the art. It may also beimportant in such a coherent system to reduce coherent optical effectsfrom the source itself, which cause position-dependence within themeasurement channel. This may be described in more detail elsewhere inthis disclosure.

In an embodiment, internal AR coating 3108 may be designed so as toprevent reflection between the lower substrate material 3102 (usuallyrelatively high index) and the liquid sample in the channel(approximately the index of water, which is relatively low). Theexternal AR coating 3104 should be designed for the atmosphere of thetool, which may be most likely air. The coatings must be designed towithstand subsequent processing and exposure to the sample andassociated fluids, without toxicity to the sample. For this purpose,very thin terminal layers may be provided on the internal AR coatings3108 and 3120.

The microfluidic features such as channel 3112 may be fabricated eitherby etching the substrate material 3108 to form a channel of specifieddepth (before AR coating), or, as shown here, formed through addition ofanother material 3110. For example, SU-8, a UV-crosslinked photoresistthat may be etched with high aspect ratio, and is known to bebiocompatible, may be spun on the bottom wafer 3102, and then patternedwith microfluidic features. The top wafer 3114 is first patterned andetched to provide ports/reservoirs and any mechanical alignment featuresindicated here by 3122. For this purpose it is desirable to use a wafermaterial where etch processes are well known, such as silicon. Internaland external AR coatings 3120 and 3118, and respectively, may bedeposited after this etch step or, if deposited beforehand, removedduring the etch process.

The top and bottom wafers may be made from different materials accordingto processing and operating requirements. For example, the top wafer maybe made from silicon, which may be readily etched using well-establishedprocesses, and may be a low-cost material in the mid-IR. Silicon,however, suffers from a number of drawbacks for mid-IR spectroscopy.First, it may have some absorption in the mid-IR which may be mitigatedby using high-purity float zone Silicon. It may also not allow forvisible or NIR light transmission, which may be desirable where visiblelight measurements may complement the infrared cellular DNA measurement.For example, visible imaging, fluorescent label measurement, or othertechniques may require a material with visible-light transmission. Insuch a case, it may be desirable to use a material such as ZnSe, BaF₂,CaF₂ or other known visible/mid-IR window as the bottom wafer, andSilicon as a top wafer.

The top wafer 3114 and bottom wafer 3102 may then be aligned and bonded.SU-8 itself may be used to establish a bond between wafers at theappropriate temperature and pressure, usually under vacuum. This may bedone with a supplemental layer or pattern of SU-8 on the top wafer, butsome groups have been successful with one-sided SU-8 bonding. The wafermay be subsequently diced into multiple microfluidic chip components.These may be individually packed into plastic carriers, or matched withcarriers in the tool, as described earlier.

FIG. 32 shows the detail of an example embodiment of a microfluidicchannel in a top view, including a measurement volume and apressure-actuated cell switch. The example may be illustrated for thecase where a mid-infrared, QCL-based optical system may be used for cellinterrogation. An incoming channel 3202 carries a core stream with cells3208 within a sheath fluid 3204 into the measurement zone 3210. Themeasurement zone may be delineated using a metal mask that allows theinfrared beam (indicated by solid line in 3212) to pass only through theaperture. In this embodiment, the aperture may be larger than the beam,so that mechanical movements do not translate into large opticalvariations. The aperture, patterned photolithographically onto themicrofluidic chip, may serve to allow alignment of the measurement beamwith the center of the channel.

As the cell passes through the measurement volume, it breaks thevisible/NIR/SWIR beam (dotted within 3212), indicating cell presence,and possibly giving some data on cell size/density. The mid-IR beam(solid line within 3212) may be absorbed by the cell according to itschemical bond constituents. The mid-IR beam may have one, two or moreindividual wavelengths acting as reference levels, or to measureconstituents other than DNA. For DNA measurement, vibrationalfingerprint regions such as the 1234 cm⁻¹ or 1087 cm⁻¹ phosphate bondvibration lines may be used to measure absorption and therefore amountof DNA present.

Based on the calculated DNA quantity, and optionally other parameters, asort destination for the cell may be determined. By default, the cellcontinues straight along the channel to the waste output (left branch).If the cell is determined to be of the sought-after type destined to the“selected” output, as it passes through the detection point 3218, thepressure actuating channels 3214 may be used to slightly offset the coreflow to the right. A visible/NIR/SWIR beam 3220 may be used in thislocation to accurately assess cell speed in the channel (from the delaybetween the measurement point and the switch actuator point). This speedmay be used to regulate the overall pressure in the system to maintainflow rates within a window, and may also be used to adjust the timing ofswitch activation relative to the cell passing through the measurementpoint.

By default the core flow may go straight 3228 into the “discard”channel. Where the switch has been actuated to select a cell, the coreflow may go to the left of the branch point 3222 and selected cells 3224may go to the “selected” out port/reservoir. Two additional optionaldetection points 3230 may be used with visible/NIR/SWIR interrogation tomonitor cells flowing into channels, so that errors may be detected andcorrected. For example, if sort failures are detected, the timing of theswitch actuation or the pressure on the system (and therefore cellvelocity) may be adjusted to provide accurate switching.

As is illustrated in the FIG. 32, cell agglomerates may be detectedeither by the visible/NIR/SWIR scattering or imaging patterns, or by theinfrared quantification. These may be sent through to the “discard”channel. Likewise, cells that may be spaced too closely within thechannel, and as a result where accurate switching may not occur, may belet flow through to the discard, depending on the system parameters.There are of course situations where very rare cells are beingcollected, in which case it is better to err on the side of switchingcells into the collection reservoir.

Through a combination of flow speed monitoring and cell timingmeasurements provided by the aforementioned optical measurement points,the dilution of the cells may be monitored. If the cells areinsufficiently diluted, core vs. sheath pressure may be varied to spacecells, or the sample itself may be diluted differently by the tool,sometimes necessitating use of a new microfluidic chip.

FIG. 33 illustrates an embodiment of the optical interrogation system ofthe present disclosure. This example may be based on two QCLs 3308 atdifferent wavelengths. For example, one QCL may be centered at the 1234cm⁻¹ symmetric phosphate bond vibration absorption peak typical of theDNA backbone. The other may be at a nearby reference wavelengthmeasuring water absorption within the channel. In this manner, waterdisplacement by the cell may be referenced out of the overallmeasurement. The mid-IR beam from the first laser here is turned by aplain mirror 3310 and then combined with the beam of the second mirrorby a dichroic filter 3312. A lens 3314, which may be a reflective orrefractive element, focuses the infrared beams onto the measurementvolume 3304 on the microfluidic chip 3302. The transmitted infraredradiation (the input beam minus the absorption of the sample in thechannel) may be transmitted via a fold mirror 3322 to a mercury cadmiumtelluride (MCT) detector 3324 that measures intensity.

The different wavelength QCLs are modulated/pulsed so their signals maybe separated in the electrical output of the MCT detector. Optionally,an additional dichroic filter and MCT may be used to separately measurea particular IR wavelength. The MCT detector may be uncooled, TE-cooled,or even liquid nitrogen cooled to achieve maximum signal-to-noise ratio.Optionally, thermal infrared detectors such as pyroelectric orbolometric detectors may be used, generally with inferiorsignal-to-noise characteristics.

In an embodiment, a reference detector, placed on the laser side of thesample, may be used to measure laser output powers in the case wherethere are significant fluctuations in laser power. In this case, a smallfraction of the beam may be sampled with a partially-reflective mirror,and a MCT or other detector used to measure power before the sample andchip absorption. This signal may be then used as a baseline formeasurements.

As described earlier, multiple signal processing techniques may be usedto establish baselines in this system and accurately measure DNA contentin cells. Most of these may be well known in the fields of cytometry andtime-series infrared absorption measurements.

A visible, NIR, or SWIR laser 3328 may be integrated into the system forthe purpose of detecting cell presence, and possibly size and density.The visible beam may be combined with the infrared beam using a dichroicfilter 3318, focused through the measurement volume, and then sent viaanother dichroic filter 3320 to a visible detector. Additional optics,such as masks, may be added into the system ahead of the detector inorder to remove the unscattered light from the measurement (achieved byblocking the zero-order light).

The chip may be constructed in a method described above so as tomaximally transmit infrared light, and reduce any optical cavityeffects.

The infrared light may also be pre-treated by devices to reducecoherence length, and further minimize coherent optical effects thatcould introduce spatial dependence in the measurement channel. One suchdevice may be described in more detail below.

FIG. 34 shows an example of the present invention using coherentanti-stokes Raman spectroscopy (CARS) to measure vibrational bondfingerprints in cells at high speed. The system consists of a pump laser3402 that may be combined with one or more slave lasers 3404 to exciteone or more bond vibrations in the cells being measured. The combinationof pump wavelength and specific slave wavelengths may serve toresonantly excite specific molecules so as to emit coherent light at ananti-Stokes frequency.

In the example shown in FIG. 34, the pump 3402 may be folded by mirror3408 and combined with two slave wavelengths from lasers 3404 usingdichroic filters 3410. The pump may be typically pulsed in synch withthe slave (one slave at a time) to maximize signal. The pump and slavewavelengths may be focused on the measurement volume 3414 and any cellscontained within it. The forward coherent Raman signal (“F-CARS”) may beseparated using a wavelength-selective filter 3418 and focused on thedetection system 3420 that may typically consist of a photomultipliertube (PMT) and associated electronics.

In addition, an epi-detected CARS (“E-CARS”) signal may be measured byseparating backward emission from the sample with a dichroic filter 3422and focusing it on a detection system 3424. Additional optical detectorsmay be used to detect the pump beam as a direct measurement ofscattering by the cells in the volume. In one example, pump beam may bepulsed alternately with each of the two slave wavelength lasers, eachcorresponding to a different vibration band or, one to a vibration bandcorresponding to DNA, and another to a reference band. Coherent Ramansignal generated by these pulses from the sample may be read out by thedetectors, and processed by the system to calculate cellular DNA asdescribed previously.

Although the laser wavelengths differ, and the readout mechanism may bedifferent, the fundamental “molecular bond fingerprint” being measuredmay be the same as when QCLs may be used to interrogate the cells.Similarly, many of the same issues come to bear in the system. Forexample, good AR coatings on the microfluidic channels may be requiredto minimize variation as a result of mechanical or temperaturevariations, and to minimize the position-dependence of the laserintensity received by the cell (and the emitted signal intensity).

FIG. 35 shows a configuration of a QCL with components to reducecoherence length, so as to minimize spatial dependence in the readout ofcell spectral measurements. The QCL 3502 may be collimated by optics3504 and then passed through a diversity of phase shifts introduced byphase plate 3508. This plate may be spun or translated mechanically soas to provide rapid changes in phase to each part of the beam, with atime constant shorter than the integration time of the system.Additional optics 3510 then focus the beam onto the chip 3512 with themeasurement volume, and the transmitted beam 3514 is relayed to detectorsystems. The “decoherence” device may be applied to multiple overlappingbeams from multiple QCLs (at different wavelengths) simultaneously. Sucha device reduces coherence length, and reduces coherent effects near thefocus of the system.

FIG. 36 shows an example of the display/user interface when the tool maybe used for cell sorting. The x-axis 3602 of the graph displayedindicates cellular DNA content. The y-axis 3604 may indicate cumulativecell count. The histogram 3608 may then generate from multiple cellularmeasurements. The tool may run a small sample of cells in order to buildup a distribution for display to the user or for fitting curves to thedistribution automatically.

In this example, a fitted distribution curve 3610 may be superimposedonto the histogram in order to indicate the spread of a particularpopulation of cells. For single-purpose tools, or for repeated protocolsperformed with a general-purpose tool, these curves may be generated andapplied automatically. For example, in a tool based on the presentinvention meant to enrich sperm cells to either X-carrying or Y-carryingpopulations, two such curves may be fit automatically onto the data, andthe user then simply selects enrichment fraction and number of cells. Aselection range 3612 indicates which range of measured DNA content maybe selected using the sorting function of the tool. This window may bemanipulated directly by the user for some applications.

Alternatively, the user may use the panel 3614 to simply specify thenumber of cells required, and the enrichment percentage desired. Thetool may then automatically calculate the optimal position for theselection window 3612 and calculate the estimated run time for the sort.

In an embodiment, where the present invention may be used for genderselection purposes, the user interface may be simplified further: a)select X- or Y-enrichment, b) select desired enrichment level, and c)select number of cells desired. The system then displays estimated runtime, and if this is within an acceptable range, the user may initiatesorting. As the sort proceeds, the projected sort time may be updatedbased on how many cells are being selected. In addition, the system mayautomatically alter the sort window 3612 in order to achieve thetargeted enrichment.

Other examples of single-purpose tools or protocols enabled by thepresent invention may include but are not limited to tools to separateout dividing cells those with double, or at least excess DNA which mayindicate cell division is in progress only or only non-dividing cells,tools to separate out cells exhibiting aneuploidy (abnormal DNA content)or only cells with “normal” DNA content.

A system may include one or more of a liquid flow cell which isspecifically designed for high-speed measurements using a mid-IR QCL, anelement for creating phase diversity in the mid-IR laser light, anoptical subsystem that ensures a narrow, focused spot is sampled in theliquid sample, an element minimizing the scattered or reflected lightthat reaches the detector, and a QCL with a rapid, low-cost, coarsetuning mechanism specifically suited to liquid- or solid-phasemeasurements. Rather than making a surface measurement of a liquid suchas that performed by existing mid-IR sampling architectures, this systemmakes a transmission measurement through the entire sample, in whichmid-IR light is transmitted directly through the sample. The systemminimizes reflections of any type, and methods may be used with thesystem to reduce contributions from scattered or reflected light.

Depending on the speed at which the absorption spectrum of the liquidmust be measured, and the QCL power available, the path through the flowchannel must likely be designed to be short because mid-IR absorption ofliquids (water, in particular) is often very high. Typically, a pathlength on the order of microns is required—for very high-speedmeasurements, 25 microns or less. A limiting factor, even in the casewhere laser power is not, is the total power absorbed by the liquid, andany temperature constraints (for example, it could complicate matterssignificantly if the liquid starts boiling in the channel).

Such a flow channel may be fabricated in one of a number of ways knownto those skilled in the art. For example, channels may be fabricated bybonding together two wafers which are transparent in the mid-IR, atleast one of which has been patterned with flow channels, either byetching into the wafer, or by adding and then patterning anothermaterial on top of the wafer. For example, SU-8, a photopatternablepolymer, may be used to pattern flow channel walls on one of the wafers,and then may be used as an adhesive to bond another wafer on top of it;finally, these wafers are diced into individual devices. Wafers mayadditionally be patterned and etched to form inlets and outlets forliquids. Channels may be narrow, or may in fact be large areas.

In some cases, the “channel” may in fact be a large liquid volumebetween two windows, and rather than the liquid flowing past thedetection point, the sample holder may be translated past a measurementsystem—with the sample holder (windows and interposing liquid space)designed as described below.

For the purpose of consistent measurements of the liquid and anycontents therein, it is important to take into account coherent andresonant optical effects that may occur in such a measurement system.Such effects may include: laser (QCL) coherence effects which cause avaried field strength and therefore absorption over the sample volume,causing position dependence within the measurement, either along theaxis of the laser (depth dependence) or laterally (x-y dependence or“speckle”); reflective or semi-reflective surfaces adjacent to thesample, which result in a spatial variation in EM field strength nearthe surface (physically, a mirror will have a field minimum at itssurface); and reflective or semi-reflective surfaces which interact toform a resonant cavity, resulting in: wavelength-dependence inabsorption due only to resonance or lack or resonance (effectively, somewavelengths will have more average passes through the liquid sample thanothers); and again, at fixed wavelength, result in a static distributionof field strength within the sample, making it unevenly sampled andposition-dependent.

The present disclosure includes a number of specific design parametersfor the liquid channel/sample holder and the optical delivery system tominimize these issues and assure higher accuracy in absolutemeasurements of the sample. These may be applied singly, or incombination, to minimize position-dependent absorption within the flowchannel of the mid-IR beam(s).

One specific design parameter may be fabricating the sample holder withwell-designed antireflection (AR) coatings on both external (air-facing)and internal (liquid-facing) surfaces. These coatings preventreflections at the interfaces, which is important both for eliminatinglocal field minima/maxima near the interfaces (which may causenon-uniform “sampling” of the liquid), and reducing resonant opticaleffects within the system. AR coatings for air interfaces are well knownfor mid-IR wavelengths. If the index of the liquid sample is close tothat of air, these may be sufficient for internal (liquid-facing)surfaces as well. However, they may require special design, including:matching of the AR coating to minimize reflection between the window(which is a material transparent to mid-IR radiation, for example ZincSelenide, Silicon, Germanium, Barium Fluoride, Calcium Fluoride, andcertain plastics with high IR transmission) and the liquid analyte(water, for example); designing the AR coating for the specificwavelength, or range of wavelengths, to be used in the spectral analysissystem; designing the AR coating for the (range of) angles of incidenceof light that will be seen by the surface, wherein the angle is acombination of the beam cone angle (most systems will be focused down tothe channel) and the angle at which the sample holder is placed relativeto the beam (see discussion below); designing the AR coating forcompatibility with the liquid analyte—since many mid-IR coatings arecomprised of materials that absorb water—either alternate materials mustbe used, or capping layers that resist water penetration must be used;and/or terminating the AR coating with layers, or post-treating the ARcoatings in order to make the surface with the appropriate hydrophilicproperties required to move fluid into the channel or cavity; possiblycoating or treating the surface in order to minimize (or, in rare cases,maximize) adherence of biological or other particles to the surface; forexample, in a flow cytometry application, ensuring that cells do notadhere to the channel walls in the measurement volume (or elsewhere);these terminal layers or treatments must of course not significantlyreduce the effectiveness of the AR coating itself.

Another specific design parameter may be angling the surfaces of thesample holder such that any reflections are rejected from the system andnot delivered to the detector, or reflected back to the laser source.The cone angle of the light focused onto the sample holder should betaken into account in this case. In addition, AR coating designs may bemodified to take into account this angle of incidence. Angling theentire channel may effectively increase the path length through theanalyte. This may be beneficial in some cases but needs to beconstrained in others where the liquid analyte (or liquid carryingparticles of interest) is highly absorptive at the target wavelength(s).The beam may be asymmetric in order to achieve higher uniformity inmeasurements—for example, in a flow application it may be desirable tohave a short axis parallel to the flow, and long axis across it in orderto have the most uniform illumination across the center of the flow—theresulting cone angles should be taken into account. In this case itwould usually be desirable to tilt around the short axis, since the coneangle along the long axis will be narrower, and therefore less tilt willbe required to ensure no reflected light is sent to the detector orlaser subsystem.

Another specific design parameter may be using nonparallel surfaces inorder to minimize resonant effects. However, the optical path throughthe liquid should be consistent in cases where particles in the liquidare to be measured, so that the attenuation due to liquid absorption isidentical regardless of particle position within the sample volume.

Another specific design parameter may be employing specific gapdistances through the channel, tuned to the interrogating wavelength(s)and the average index of the liquid analyte or liquid carrier plussample particles. A channel thickness (or gap) may be specificallynon-resonant at the wavelength of interest, meaning that the gap timesthe index of the liquid contained in it are a non-quarter wave multipleof the interrogating wavelength. This prevents constructive ordestructive interference effects to the maximum extent possible,reducing buildup of resonances in the cavity, and in this mannerminimizing spatially dependent absorption in the system.

The system is focused on enabling high-speed liquid-phase (or solidparticles/cells within a liquid medium) spectral measurements. Liquid-and solid-phase absorption lines in the mid-IR are far broader (severalwavenumbers at a minimum) than gas-phase absorption features. As aresult, it is acceptable to have broad linewidth, but at the same time,it is necessary, to effectively have a wide tuning range in order toreach one or more absorption peaks of interest, as well as referencepoints in the spectrum which are used to establish a baseline. For thesake of speed, this tuning must be performed very rapidly over thisbroad range. So the requirements on the QCL subsystem are very differentthan for gas-phase spectroscopy. The system is described as separatedelements in a system, as some applications will benefit from thesimplicity of having a separate microfluidic element—that may bedisposed of after use in some applications, or at least swapped outperiodically.

One of several designs for the QCL subsystem may be used in the presentdisclosure. One design is discrete fixed-wavelength QCLs that areoptically multiplexed. In this design, individually packaged QCLs areused. They may be either distributed feedback (DFB) or external cavityFabry-Perot (FP) type (where wavelength is set in the external cavity).The outputs of the lasers are collimated, and are combined with oneanother, typically using either dichroic or bandpass filters (which, forexample, reflect one wavelength and transmit others). If power is not anissue, semi-transmissive mirrors may be used to multiplex the beams.These lasers may then be pulsed in rapid succession to measureabsorption at different peak and reference wavelengths of the liquid inthe microchannel. Alternatively, if the QCLs are operated in continuouswave (CW) mode, they may be modulated with different frequencies andtheir signals demultiplexed electronically after detection by a mid-IRdetector (after passing through the liquid sample). Opticaldemultiplexing to multiple detectors is also a possibility, where thehighest signal-to-noise ratio is absolutely necessary. The advantage ofmultiple discrete QCLs in the present invention is that they are morereadily available from suppliers today, and that they may be changedrelatively easily (for example, if systems with different chemicaltargets are being built). In addition, they may span very broadwavelength ranges, whereas the tunable solutions described hereafterwill cover a relatively narrow range of wavelengths (and therefore mayrequire the use of several tunable QCL subsystems within the presentinvention).

Another design is the QCL array-on-chip. This design consists of anarray of DFB lasers fabricated on a single chip. This means a singlegrowth design is used, but gratings that set laser wavelength areindividually patterned to result in different center wavelengths. Thepotential advantage of this architecture is the lower cost of packaging,cooling elements, and associated elements. It also opens the potentialfor a very compact system that may be rapidly switched from onewavelength to another simply by electronically switching between lasersin the array. The array may be produced with regular-spaced wavelengthover the interval of interest (which covers one or more absorption peaksin the liquid sample, and perhaps one or more reference absorptionmeasurements). Wavelengths may be spaced apart at wider intervals, andeven at irregular intervals. For example, the array could contain 4discrete and unevenly-space wavelengths over a frequency interval of 200cm−1, corresponding to peaks and references. For example, if cellularDNA in live cells flowing through the liquid channel is to bequantified, and therefore the characteristic symmetric phosphate bondpeak at 1087 cm−1 is to be measured (phosphate is an element of the DNAbackbone), three closely-spaced wavelengths at 1075 cm−1, 1087 cm−1 and1099 cm−1 could be employed to measure absorption peak absolute heightand “shape,” and another wavelength at 1055 cm−1, corresponding to anearby absorption minimum (and therefore potentially good referencelevel) could be added. In this manner, a single chip containing allrelevant wavelengths may be fabricated and packaged with minimum sizeand cost. The beams from the lasers on the common chip may be deliveredin at least two ways to the microfluidic channel: 1) A grating may beused to redirect individual wavelengths in a manner such that a single,overlapping collimated beam is formed. The coincident beams may then befocused onto the microfluidic channel where they are absorbed by theanalyte, and then relayed to a detector. Note that for irregularlyspaced wavelengths, laser diodes may have to be spaced accordingly onthe chip in order to have a diffraction grating accurately redirect thebeams into a single overlapping beam. Or 2) The plurality of lasers maybe imaged directly onto the microfluidic channel using appropriateoptics. In this case, an array of points corresponding to the array oflasers will be projected onto the microfluidic channel. In a 1:1 imagingsetup, for example, if the DFBs are patterned 20 microns apart on thechip, a series of spots 20 microns apart will be sampled in the liquidchannel. For example, these could be oriented along the direction offlow of a microchannel, and the liquid and anything being carried by theliquid would be sampled sequentially by the beams emanating from theseries of lasers on the chip. The wavelengths of the lasers could inthis case be in any pattern; for example they could simply be analternating array of two wavelengths (signal and reference wavelengths).In other configurations a larger number of wavelengths could be used tobuild up a rough spectrum. If configured in this way in a system whereparticles such as cells are run through the liquid channel,appropriately spaced, the lasers could in theory be run in CW mode, andsignal processing could be used to extract the absorption levels. Inmost cases, however, individual lasers will still be pulsed in rapidsuccession, or modulated at different frequencies to provide for easyelectronic separation after detection by a mid-IR detector. The spotsfrom the array may be imaged such that they are not entirely parallel tothe microfluidic channel. They could be oriented in a diagonal manner inorder to give some lateral resolution to the system. Such lateralresolution within the flow channel could be used for example to measureposition of cells within the channel as they flow by (to compensate forany known/calibrated spatially-dependent detection nonuniformities), or,for example, to measure concentrations across a flow withnon-uniformities, from either differential velocities (center vs. edge),or because two liquids are mixed upstream in a largely laminar flow.

Another design may a QCL that rapidly tunes to discrete wavelengths. Asdiscussed earlier, continuous or fine-stepped tuning is not required forthe liquid spectroscopy application. High speed is a requirement. Arapid tuning mechanism that samples sparse wavelengths would be ideal.One tuning mechanism that lends itself to potentially rapid, discrete,controllable, low-cost tuning is a Vernier tuning arrangement in theexternal cavity of a FP QCL. In such an architecture, thermal tuning(for example) can be used to achieve much faster, broader tuning thanwould otherwise be possible. Such architectures are well known and havebeen used to build telecommunications lasers in the 1.5 micron range. Wepropose that such an external cavity architecture for a mid-IR (oreventually THz) QCL, integrated into a system with a fluid microchannelin which absorption is measured, would be a potentially ideal solutionfor high-speed liquid spectroscopy. In the Vernier filter configuration,two Fabry-Perot resonant elements are used in the external cavity,designed with free spectral ranges (FSR) that are close but not equal.By slightly tuning one or both of these elements, different transmissionpeaks will coincide. In this manner, the portion of the gain spectrum ofthe QCL that is amplified is selected. The advantage is that withrelatively little input signal to the Fabry-Perot cavities (in the formof temperature in thermo-optically tuned systems, or voltage inelectrostatically-tuned systems), large hops in wavelength may beachieved. Furthermore, the wavelength settings themselves are relativelywell controlled since the modes of the Fabry-Perot cavities are known.The spacing and centers of these wavelengths may in fact be optimizedwithin the present invention to provide the most efficient measurementof the liquid or liquid-suspended analyte. For example, the spacing ofemission peaks could be configured to fall on the absorption peak ofinterest and on a reference point only for maximum efficiency. With theaddition of a third filter to the external cavity of the QCL, one couldsample a few wavelengths around the absorption peak, then a fewwavelengths at one or two reference points.

More conventional tunable QCLs (such as those tuned by piezo-actuatedexternal gratings) may also be used in the context of the presentinvention, in conjunction with other elements described herein, so longas they provide the required tuning speed.

Unlike with hot filament blackbody (“globar”) sources used in FTIRinstruments, QCLs are inherently coherent optical devices, and a numberof potential complexities arise as a result. Coherent effects in imagingsystems are well known (“laser speckle”) and arise from constructive ordestructive interference of the laser light returning from variouspoints in the sample. We wish to avoid such effects, to the extentpossible. A few elements described below may be employed to ensure thatcoherent effects—particularly spatially-dependent effects—are minimized.One element involves optical apertures—optical apertures through whichthe interrogating beam is focused may serve to reduce optical effectsfrom the laser, and provide higher consistency in measurements. Anaperture on the QCL side of the system (before the sample) may be usedto “clean up” the beam from the QCL, and therefore deliver the minimumspot size onto the sample in the microfluidic channel or cavity. In thisconfiguration, lenses are used at the output of the QCL to focus mid-IRlight through an aperture of roughly 20 microns or less (approximatelythe desired spot size on the sample). This aperture will be imaged ontothe sample, and ensure that a “clean,” small spot is sampling theliquid. An aperture on the detector side of the system (after the beampasses through the sample) may be used again to “clean up” the beam—thistime also removing any scattered light from the measurement.Combinations of scattered and directly transmitted light in a coherentsystem could cause unwanted effects. In addition, elimination ofscattered light from the measurement ensures that only light that haspassed more or less directly though the sample (and therefore reflectiveof Beers law absorption) will be measured in the system.

Another element involves phase scramblers. Phase scramblers may be usedon the input (QCL) side of the system in order to de-cohere the lightimpinging on the sample. Scramblers typically rapidly change opticalphase across a beam, on a time scale some multiple shorter than themeasurement time (for a single event). In this manner, coherent effectssuch as those described above are effectively “averaged out” over anumber of states. One example of a phase scambler is a transmissive discthat has been etched with a pseudo-random pattern of fields withdiffering phase delays (by virtue of material index and thicknessdifferences). This disc is the spun at an angular rate sufficient to“scramble” phases over a single measurement, and the beam from the laseris sent through the disc before it is focused onto the sample. Wepropose such a phase scrambler, designed for the mid-IR, combined with amid-IR QCL, for microscopic spectroscopy applications in the mid-IR suchas the ones described herein.

The present disclosure may be applied to, and form the core of, multipletypes of systems that rely on rapid liquid-phase spectral measurements.

One such system is a fast reaction monitoring system. In such systems,very small volumes of reagent are used in a microchannel or microcavity,and the ensuing reaction is measured. The advantage of the presentsystem is its capability to measure chemical concentrations, or evenchanges in chemical configuration (shape, folding, etc.) at high speedin liquid phase, and producing accurate results on an absolute scale(rather than just a time series of relative measurements). Suchcapability could be used for medical diagnostics, environmental tests,or combinatorial chemistry done at large scale on a chip.

Another such system is a high-throughput screening system. Manytechnologies have been devised to measure changes in cellular behaviorin response to potential drugs. Most of these sample the cellsuperficially (for example, with surface-oriented optical techniques),and/or do not have the ability to directly measure key biochemicalconcentrations within the cell. The present invention enables suchmeasurements to be made, consistently, in the native liquid environment,and at high speed. High speed may enable either very rapid successivemeasurements in a single cell, or will allow the system to step rapidlyover a large number of wells in which cells have been placed withcompounds. The ability to sample small volumes of liquid, containingcells, in a manner not dependent on cell position within the volume—andto do so accurately and quickly—is an advantage of the presentdisclosure.

Another such system is for high-speed cell classification. Emergingtechnologies that trap cells in a microfluidic cavity using structuredand/or coated surfaces often require follow-up measurement and screeningof cells to classify them. Often extremely rare cell events are ofinterest. For example, circulating tumor cells, or embryonic cellswithin the mother's bloodstream are typically 1 in a billion or less infrequency. Microfluidic structures may serve to enrich these to 1 in1,000 or 1 in 10,000 in frequency. Subsequently, the trapped cells(which include many white blood cells, for example), must be screened inorder to find true occurrences of the rare cells. The cells identifiedmay then be extracted for further analysis. The present invention may beused to build a system for performing these screens rapidly on apopulation of cells that are trapped within a microfluidic cavity. Knownbiochemical markers established with FTIR or Raman spectroscopy inresearch studies may be interrogated using the present invention toaccurately and rapidly screen and classify cells. Stem cell cultures maybe measured in a similar manner to determine state of differentiation,while in a microfluidic cavity.

Another such system may be rapid cell counting/classification in a flowsystem. The present disclosure may be embodied in a format where asuspension of cells, or bodily fluid containing cells, flows through amicrofluidic channel, and cells are interrogated as they pass throughthe mid-IR beam. The absorption of one or more wavelengths is measuredby the system, and the cell is classified, or measurements are directlydisplayed to the user as a histogram. For example, cellular DNAmeasurement for cell cycle characterization may be done. Distribution ofDNA quantity in a cell sample is indicative of how quickly cells in thesample are dividing and therefore multiplying. Cells with twice the“normal” DNA are in the process of dividing. In another example,cellular DNA measurement for aneuploidy detection may be done. Unusuallevels of DNA are often an indicator for cancer. Detection of an unusualdistribution of DNA within a sample can be a strong indicator that asample is cancerous. In another example, blood cell counts may bemeasured. A complete blood count, including counts of all white bloodcell types, may be possible using embodiments of the present disclosure,without the use of fluorescent labeling or other preparation. Cellswould be classified using a combination of mid-IR wavelengths,potentially in combination with visible-light detection schemes. Besidesindividual cell measurements and counts, the liquid content of the bloodplasma itself could be analyzed using the same system. In anotherexample, an analysis of semen could be performed using embodiments ofthe present disclosure. Sperm cells could be counted and potentiallycharacterized (for proper DNA packaging and chromosome count, gender);other cell counts could be determined (white blood cells); seminal fluidcharacteristics measured.

Another such system may be a cell sorting system. Well-known sortingmechanisms may be added to the cell measurement systems above in orderto sort cells into distinct populations based on the measurementsdescribed. For example, sperm cells may be sorted for gender, cellsexhibiting abnormal DNA count (potential marker for cancer) may beselected for analysis, cells that are actively dividing may be selectedto create a sample with high viability, and cells may be sorted based onother biochemical markers; for instance in development of biofuels,cells that are determined to be producing a large amount of the targetchemical may be identified spectroscopically without destroying thecell, and cycled back into the cell culture, while “less successful”cells are disposed.

Another such system may be for embryo scoring. In in-vitro fertilizationprocedures, it is strongly desirable to implant the minimum number ofembryos required for a successful pregnancy. To this end, there issignificant research ongoing into “scoring” embryos for viability, basedon biochemical and/or morphological changes in early development. Thereis research that indicates biochemical concentrations could be a keyindicator for viability. Embodiments of the present disclosure could beused to interrogate and score embryos, in a liquid environment, withoutthe use of dyes or labels, and using only very low photon energy light.The ability to rapidly measure spectra, and potentially track spectraover early development, could be a significant advance in the ability toscore and select embryos for implantation.

Another such system includes a gas monitoring system. In some cases,monitoring gas flows directly (sometimes done using mid-IR spectroscopy)is not practical. In such cases, it may be possible to flow gas over orthrough a liquid stream that reacts with the target compound in the gas.The liquid stream flows through a microfluidic channel where it is inturn interrogated using the present invention. We propose this system asa whole as an effective, compact, rapid manner of monitoring some gascompositions—for example, detecting trace impurities in gases, ordetecting biological or chemical agents in air. Similarly, a liquidsurface may be exposed to air or another gas flow for a specifiedduration, and then the liquid, together with any particles trapped overthat duration, analyzed using the present invention.

Another such system may be a solid sampling system. Sampling of solidsby infrared spectroscopy is a long-standing challenge. There is verylittle optical penetration into most solids by mid-IR. As a result,surface techniques such as ATR must be used, which are often limited bysurface layers or texture. The liquid spectroscopy system describedherein may be used in an embodiment where a solid is sampled bymechanically fracturing/grinding it into very small particles, filteringthese particles to ensure a reasonable range of diameters/shapes, andthen suspending them in liquid for subsequent measurement. Measurementcould be performed in a cavity where the solid particles are dispersedacross an area (or a line), or with liquid that flows through a channelthrough a small measurement volume. According to the designs laid outabove, the liquid and suspended solids are the measured using mid-IRtransmission. In one embodiment, one mid-IR wavelength may be used tomeasure water absorption, in a range where the solid of interest doesnot absorb strongly in the mid-IR. As a particle moves through theoptical detection area, its volume may be estimated by the decrease inwater-line absorption. At the same time (or in rapid succession), theabsorption of the solid particle at the target wavelength is measured.It may then be normalized for particle volume in order to calculate thechemical composition of the particle. An example embodiment is anenvironmental sampling tool. A tool with a small drill is constructed todrill into a layer suspected of being asbestos. A small amount of liquidis injected, and then a capillary-type tube is used to sample thisliquid with any suspended solid. This liquid is then interrogated at theappropriate wavelengths to determined chemical composition and structureto signal whether the substance is asbestos. Pharmaceutical purityinspection is another example.

Another such system may be an emulsion measurement system (oil in water;water in oil).

FIG. 37 shows a sample embodiment of the present invention that mayinclude elements to minimize spot size on the sample, and to rejectscattered light from the sample. A QCL subsystem 3702 which may consistof one or more mixed or tunable QCLs in the mid-IR or THz range may becollimated and then refocused by lenses 3704 and 3708 into an aperture3710 that serves to “clean” the beam from the QCL and ensure aminimum-size, Gaussian beam profile at the sample. Lenses 3712 and 3714and may be then used to refocus the beam with minimum spot size onto thefluid microchannel in the sample holder 3718. The sample chamber may beshown here at an angle to ensure any stray reflections are notpropagated to the detector, and to reduce any resonant optical effects.The flow of the liquid shown here by arrows may be a sample that may betransported through a channel, through the measurement volume onto whichthe QCL-originated beam is focused. Lenses 3720 and 3722 collect thelight that may be transmitted through the liquid in the sample holder,and refocus it onto another aperture 3724 which may serve to blockscattered light from the sample or sample holder, and restrict, to theextent possible, light reaching the detector to directly-transmittedradiation, thereby giving the best possible absorption measurement forthe liquid sample and any suspended particles/cells. Lenses 3728 and3730 deliver the light to a detector 3732, for example a mercury cadmiumtelluride (MCT) detector for the mid-IR (which may be cooled bythermoelectric elements or liquid nitrogen).

FIG. 38 shows several sample configurations used by others that maysuffer from surface effects that the present invention is specificallydesigned to avoid. FIG. 38a shows an attenuated total reflectance ATRconfiguration often used to measure liquids or solids using FourierTransform Infrared (FTIR) spectroscopy in the mid-IR. Here it is shownin contact with a liquid micro channel showing flow velocity in thecross-section. As is usually the case in such systems, velocity near theinterface may be very low. Also shown is the limited penetration of theevanescent field from the ATR prism into the liquid flow. The advantageof using ATR in conventional FTIR systems is that even when liquidabsorption is very high, very little light may be absorbed in thisconfiguration. The strong disadvantage obvious from this figure may bethe limited depth of penetration into the core of the flow. FIG. 38bshows a more recent configuration used by a number of groups, which maybe similar but uses plasmonic layers (patterned metal conductive layers)to enhance absorption. This may strongly enhance absorption signaturesof samples in direct contact with the plasmonic filter. Again, however,the mid-IR field may have very limited penetration into the liquid. Formeasurements of stationary cells adhered to the substrate as shown byone of the biological cells in this example, this may allow time seriesmeasurements. However, for high-speed interrogation of cells passingthrough a liquid microchannel, this may not be an appropriatearchitecture, because of the strong depth dependence and limited depthof the absorption. FIG. 38c shows a true transmission architecture thatmay have been used for mid-IR measurements by several groups. In this“transflection” architecture, mid-IR light passes through the sample,may be reflected by a mid-IR reflective substrate (which may betransmissive in the visible), and then makes a second pass through thesample before proceeding to the detector. Holman et al described an“open channel” measurement based on this architecture, where there is notop window rather it is open and liquid flows for a limited distanceover the reflective substrate. The advantage of this may be improvedtransmission and more simple construction. The strong disadvantage maybe that any variability in liquid layer thickness may result in largeapparent changes in sample absorption. A more substantial problem withthe transflection architecture, however, derives from interferenceeffects resulting from the reflective substrate. For example, a cellvery close to the reflective surface, where the electric field mustnecessarily fall to a low level, may absorb relatively little mid-IRlight. Conversely, a cell at a certain distance from the substrate willabsorb a maximum amount of light. This dependence on depth will causevariations in signal that will be difficult to compensate. In addition,in this architecture it may be difficult to distinguish which light maybe reflected by the sample, scattered by the sample, or transmittedthrough the sample. The present invention may seek to remove most of theissues with these architectures.

FIG. 39 shows an example embodiment of a microfluidic channel describedin the present invention. A fluidic channel 3902 may carry fluid and, inthis example, biological cells 3904. The channel may be fabricatedbetween two mid-IR transparent windows 3908, whose thickness may notshown to scale (typically, the channel will have a thickness on theorder of 10's of microns, and the windows will have thickness on theorder of 100's of microns). The windows may be fabricated from mid-IRcompatible materials such as Germanium, Silicon, ZnSe, CaF₂, and thelike. The windows may be antireflection coated, with a coating toprevent reflection at the air interface 3910, and another coating on theinternal surface 3912 tuned to prevent reflection at the liquidinterface. A mid-IR beam carrying one or more mid-IR wavelengths 3918may be then focused onto a sample volume in the fluidic channel.Indicated here may be the average path length 3914 which may also betuned to minimize any resonant effects in the fluidic cavity. In thisexample, the beam may be brought in at an angle to guide any strayreflections away from the detector, and to minimize any resonant effectsin the fluidic channel. As cells pass through the measurement volume,absorption changes at one or more mid-IR wavelength (which may be usedserially). The system may detect the signals corresponding to theabsorption levels, remove background levels, and calculate the chemicalconcentration of one or more cellular constituents. For example, DNAlevels may be interrogated using the present system. The microfluidicchannel may be fabricated on a disposable microfluidic chip and carrierso as to prevent contamination. An alternative configuration does notuse a flowing channel, but rather a 2-dimensional planar cavity in whichmany cells are immobilized. The chip may then be translated in x- andy-directions, possibly guided by a visible-light system that identifiescandidate cell locations, and mid-IR is used to interrogate cells. Forexample, in circulating tumor cell (CTC) applications, a 2D microfluidicpattern may be used to trap rare CTCs in blood. The present inventionmay be used to scan the trapped cells, and detect actual CTCs amongwhite blood cells and other particles trapped in the array.

FIG. 40 shows how a microfluidic cavity may be optimized to reduceresonant optical effects, such that as much position dependence aspossible is taken out of the QCL-based fluidic measurement system. FIG.40a shows a microfluidic gap which may be an even quarter wave multipleof the interrogating wavelength, which may be a peak resonance. Thediagram on the left shows electric field, the optical intensity may beshown to the right. As may be seen, there may be a strong peak near thecenter of the fluidic gap. In general it may be desired to avoid suchstrong spatial dependence, and have more uniform sampling of theabsorption in the liquid (this may be particularly important when asuspension is being measured, where particles may be positioned atvarious heights within the channel). FIG. 40b shows the preferredconfiguration, where a gap may be used that may not be resonant at theinterrogating wavelength(s), and ensures more uniform sampling of thecontents of the fluidic channel. This configuration may of course beused in conjunction with AR coatings and angling as described.

FIG. 41 shows an embodiment of the present invention based on aVernier-tuned external cavity quantum cascade laser. This type of laser,which may be a well known architecture, may be ideal for certain liquidspectroscopy applications, because these applications require only roughbut fast tuning to a relatively small number of emission peaks within aparticular wavelength range. Several of these lasers may be used tointerrogate a liquid and/or suspended solids within a particular system.A gain medium 4102 emits mid-IR light on its rear (low-reflectivity)facet, which may be collimated by lens 4104, and then passes through twoetalons 4108 and 4110. The free spectral ranges of these etalons may beslightly different, so that only one set of transmission peaks coincidesover the gain range of the gain block. The etalon wavelengths may betuned by thermal or mechanical means. Importantly, only slight tuningmay be required to tune rapidly over a wide range, in large stepsparticularly compatible with liquid measurements where absorption peaksmay be broad. A rear mirror 4112 returns the beam back through theetalons to the gain medium. Light emitted through the high-reflectivefront facet may be collimated and refocused by lenses 4114 and 4118.Note a subsystem for “cleanup” of the beam employing additional lensesand a small aperture may be used to achieve minimal spot size. The beammay be then focused onto the liquid sample holder 4120 including amicrochannel or microcavity. The transmitted light may be then focusedonto a mid-IR detector 4124 using lens(es) 4122.

FIG. 42, taken from U.S. Pat. No. 6,853,654, further illustrates theVernier tuning mechanism proposed for high-speed mid-IR liquidspectroscopy as part of the present invention. It shows the transmissionspectra of two etalons used to tune the laser, their different FSRs, andhow they coincide on a single peak.

FIG. 43 shows the examples of multiple wavelengths used in the presentinvention. The individual wavelengths may be produced by a tunable QCL(for example, the Vernier configuration specifically described herein),individually-packaged fixed-wavelength QCLs, or a monolithic QCL arrayon a chip, delivered either as individual beams, or combined into asingle spot. For the purposes of this figure, the horizontal axisrepresents mid-IR frequency, and vertical axis represents absorbance.FIG. 43a shows a configuration where an absorption spectrum may bemeasured at an absorption peak of interest 4302, with three points beingmeasured on the peak. This allows the shape derivative, or secondderivative to be measured as well as absolute absorption. Secondderivative may be often used to measure an absorption peak in thepresence of broad background signals. In addition, a local absorptionminima 4304 may be interrogated by one of the system wavelengths. Thismay allow a measurement of the background absorption level, forinstance, of the liquid medium delivering cells or particulates into themeasurement volume. FIG. 43b shows a more minimal configuration, whereinonly a peak absorption wavelength 4308 and a reference wavelength 4310may be sampled.

FIG. 44 shows another example embodiment of the present invention. Asingle-chip QCL array 4402 may contain multiple QCLs at multiplewavelengths. These may be collimated by lens 4404 and then treated usinga rapidly-moving phase delay element 4408 in order to reduce coherencein the system, as described earlier. Another lens 4410 may focus thebeams onto the microfluidic channel 4412. In this example, the laserarray may be imaged onto the microchannel such that a series of volumesmay be illuminated along the axis of flow. The transmitted portions ofthe beams may be then delivered to a mid-IR detector 4418 via one ormore lenses 4414. In this configuration, different points along thechannel may be sampled by each wavelength/laser. For example, in acytometry system for measuring biological cells, a cell will passthrough one beam after the next, causing different signals on the mid-IRdetector. The changes in signal as the cell passes may then beprocessed, and chemical concentrations calculated. The individual lasersmay be pulsed sequentially such that individual signals are easilyresolved, they may be modulated with different frequencies or they maybe used in continuous mode, and location of the cell inferred from thepattern of signals experienced as it may move through the channel. In asystem using CW lasers, analog or digital differentiation may be used toisolate signals corresponding to absorption by a cell moving through thevolume. The potential advantage of CW lasers, besides total opticalpower, is stability. CW lasers may be sufficiently stable to make aseries of fast, referenced measurements as one cell passes through themeasurement volume with reference power levels read out before and afterthe cell is in the volume whereas pulsed lasers may vary from pulse topulse and require an additional detector in the system to reference QCLpower.

One of the problems raised in mid-IR microspectroscopy is that ofscattering. Mie scattering is dominant when the particles in the pathare on the order of the interrogating wavelength. The magnitude andangle of scattering is determined by size of particles and index ofparticles relative to the medium. Described is a high speed particlemeasurement system for measuring the chemical composition/content ofparticles in the mid-IR using QCLs emitting at a few discretewavelengths and optical system architectures to mitigate or harnessscattering effects for the purpose of making these measurements.

Scattering is generally quite large in the visible regime, as the probewavelengths used are small relative to the cellular dimensions. Inaddition, measurements made in this regime are generally dependent onwavelength only insofar as scattering is dependent on wavelengths vs.feature size; refractive indices are relatively constant over wideranges within the visible.

In the mid-IR, refractive indices of cellular components may vary quiterapidly: in the “fingerprint region” for compounds, molecular bondsvibrate at frequencies corresponding to the mid-IR. At thesefrequencies, molecules absorb light, resulting in absorption bands. Ingases, these absorption lines are generally extremely narrow. In liquidand solid substances, these bands are broadened.

Because there are localized absorption bands, corresponding to raisedimaginary components in the complex refractive index, there isnecessarily a local fluctuation in the real refractive index of thecompound, as may be demonstrated or calculated from the Kramers-Kronigrelation between the real and imaginary components of refractive index.

As a result, when designing systems for measuring particle spectroscopicproperties in the mid-IR, these local refractive index fluctuationsshould be considered, and their effect on scattering as a function ofwavelength. In some cases, system design may be optimized to minimizeeffects of this scattering. In others, references may be used tocharacterize the scattering intensity and compensate for it in anabsorption measurement. Finally, in certain instances, scattering andits strong wavelength dependence near absorption peaks may be harnessedto perform a measurement.

FIG. 45 shows a schematic example of a molecular absorption peak in themid-IR. FIG. 45a shows the absorption of the particle (composed in partof the target molecule) and the medium in which it is measured, both asa function of optical frequency. FIG. 45b shows the derived realrefractive index of the particle and the medium. As shown in FIG. 45b ,there is a fluctuation in particle refractive index around the center ofthe absorption peak. The particle has a baseline refractive index(“index at infinity”) that may be different than that of the medium. Forcertain applications it may be desirable to change the medium to have arefractive index closer (less scatter) or further (more scatter) fromthe particle. If a straight absorption measurement is desired, it isdesirable to reduce index mismatch so as to minimize the effect ofscatter. On the other hand, if a scattering-based measurement ofparticle size, for example, is desired, then it may be helpful to lowerthe medium index relative to the particle index.

FIG. 45b also indicates three frequencies, the center frequency for theabsorption band ω0, a high index point ω−, and low index point w+. Themaximum and minimum scattering may occur roughly around these points(scattering will generally rise with frequency). In building adiscrete-wavelength measurement system for particles or cells, it may becrucial to select signal and reference wavelengths while taking intoaccount wavelength-related scattering. Further, in order to minimizescattering losses, it may be desirable to shift these measurementstowards low-scatter (low index differential) regions of the spectrum.

Another factor to optimize in wavelength selection is complex index(absorption) of the target. In an embodiment where absorption is veryweak, local resonant scattering is also weak. Therefore, it may beoptimal to probe the particle at the absorption peak. Even if there arechanges in shape or orientation of the particle, the effects onmeasurement may be weak, as there is minimal self-shading. However, ifabsorption is very strong, not only will there be strong resonantscattering effects as a function of wavelength; there is also thepotential for strong orientation dependence in the measurement. This ispartly as a result of scattering dependence on orientation (notaccounted for in Mie scattering models, which assume sphericalparticles) a short path through a large cross-section may be differentthan long path through small cross-section. However, ignoringscattering, the pure absorption signal of a non-spherical particlebecomes orientation-dependent when absorption is high. This is a resultof the exponential decay of light intensity on the path through theparticle vs. the linear change with cross-sectional area. For largeexponent factors (absorptivities) there may be a stronger mismatch asthe particle rotates relative to the interrogating beam. Therefore, itmay be desirable in many cases to select off-peak wavelengths forabsorption measurements.

FIG. 46 shows a generalized system diagram for measurement of mid-IRabsorption of a particle of cell, including but not limited to livingcells. A QCL 4602 is the optical source for the system. In the contextof the present invention, QCL may be mid-IR or THz-emitting quantumcascade laser, or multiplicity of lasers. The QCLs may be fixed inwavelength or tunable with one of a number of known tuning mechanisms.In the case where a multiplicity of QCLs are used, they may be focusedonto the same measurement volume, or different volumes, and then theparticle may translate across volumes (or vice versa, with the systemtranslating across particles) and absorption/scattering from differentQCL sources measured sequentially.

As shown in FIG. 46, light emitted from the QCL 4604 is delivered to theparticle being measured 4608. Light passing directly through theparticle 4610, with some fraction absorbed according to the wavelengthand the molecular composition of the particle, may be relayed to anappropriate detector 4612. In this embodiment of absorption measurement,where gas or uniform mixtures are being measured, it may sufficient.However, in other embodiments where particles are measured (such ascells in a suspension, an emulsion, solid particles in a liquid stream,or indeed liquid droplets in air), it may be insufficient. In suchembodiments, particularly where the particles size approaches thewavelength, significant scattering may occur in an angle-dependentmanner, depicted here by scattered rays 4614. Unless the system isdesigned to capture or compensate for this scattered light, it mayobtain misleading absorption measurements for the particle or particles.

FIG. 47 shows one approach to remedying the scattering problem. QCL(s)4702 are focused using optics 4704 to form an input beam 4708 with aspecific angle, focused onto a measurement volume containing particle(s)4710. On the output side, both directly-transmitted (and shallowscatter) light 4712 as well as scattered light within a certain angle4714 are collected using a high NA lens as part of the collection optics4718. With a sufficiently large differential between the input beammaximum angle and the output collection angle, scattering losses may bereduced. For a system with a small enough refractive index differentialand particle volumes vs. wavelength, this may be sufficient to removemost scattering effects from the absorption measurement. As describedearlier, probe wavelengths may be optimized as well to ensure no excessscattering losses (at least to the level required by the accuracyrequired in the system).

FIG. 48 shows an alternative architecture where scattered light ismeasured directly. Scattered light measurement may then be used in anumber of ways. First, it may be used to estimate the total amount ofscattered light in the system, including lost light, to correct for thisloss in the output of the system. Second, it may be used to estimateparticle parameters including index and volume, together with the directabsorption measurement(s). Third, it may provide a gating signal forabsorption measurements, because it provides a positive signal on a zerobackground, while the absorption measurement may be a small delta on abright background.

The components of this embodiment include: QCL(s) 4802 are focused usinginput optics 4804 into an input beam 4808 with a smaller angle than theoutput collection angle, onto particle(s) 4810 in the measurementvolume. A high NA collection lens 4812 is used to collect both thetransmitted light, as well as scattered light within a given angle. Somescattered light at high angles 4814 will be lost in the system. Afterthe collection lens, an annular mirror 4818 is used to divert thescattered light portion through a focusing lens 4820 onto one detector4822. This “scattered light” detector 4822 measures primarily scatteringfrom the particle(s). The signal from this detector may be used asdescribed above. The directly-transmitted or small angle scattered lightis focused by a focusing lens 4824 onto the direct transmission detector4828 which may be the primary absorption detector.

In this embodiment, the system may operate with a multiplicity ofwavelengths to determine chemical concentrations and other particleattributes (size). With multiple QCL wavelengths in this architecture,both absorption and scattering may be measured at multiple points on thecurves shown in example form in FIG. 45. By measuring scattering at aknown angle at multiple wavelengths, when refractive index varieslocally with wavelength as it does in resonant mid-IR measurements, itmay be possible to accurately determine particle properties fromknown-angle, multi-wavelength measurements—while determining specificchemical concentration as well. This is a novel capability that is notpresent in visible or near-visible light conventional flow cytometers,and has not been explored in mid-IR or THz systems because of the lackof powerful, wavelength-matched, brilliant sources. QCLs fill this gapfor measurement of cells and other particles in liquid streams.

Furthermore, the present invention may be used in a system that isdifferentiating populations of particles, such as cells, the outputs ofthe scattered light detector 4822 and direct-transmission detector 4828at multiple wavelengths may be used with existing algorithms thatoptimize separation between populations. In a sorting-type system, theparameters may be refined continuously to maximize separability ofpopulations. The QCL-based, absorption resonance-tuned infrared forwardtransmission and scattering system offers multiple capabilities in avariety of applications, and may be tuned accordingly.

FIG. 49 shows an embodiment that has the potential to use QCLs tomeasure particle size and chemical concentration through scattered lightonly. In this embodiment, the system works even when particles arestrongly absorbing at certain resonant wavelengths (for example, whenDNA is very densely packed in the nucleus of a cell). It may also havethe potential advantage that all signals are zero-based; in other words,when no particle is based, sensor readings are close to zero (even withthe QCL sources powered up); particles entering the volume causepositive readings on the scatter detectors. The absolute and relativereadings on scattered light detectors 4924 at one or more wavelengthsmay be used to determine both particle size and chemical composition (orat least concentration of one target compound within the particle).

In FIG. 49, the components of the embodiment include two QCL sources4902 and 4910 to illustrate schematically how their signals are combinedusing collimating optics 4904 and 4912, and a beam combiner 4908 whichis optimally a dichroic thin film interference filter. The combinedbeams are focused using lens 4914 onto the particle(s) 4918 in themeasurement volume. Light is scattered in a wavelength- andangle-dependent manner, some of which is captured by collection lens4920. A series of spatial filters 4922 (in this case annular mirrors)are then used to select ranges of scattering angles for detection bydetectors 4924. There are a number of ways to achieve thisconfiguration, including detectors that are segmented, blocking orreflecting filters, etc. Optionally, direct transmission is measuredwith another detector 4928.

While this example is shown to have symmetry around the output beam (all360 degrees around an annular section is diverted into a single scatterdetector), the system may be further refined to have horizontal andvertical scatter detectors, or more detectors arranged in a circularmanner around the beam. This would allow more complex cell shapes to beaccounted for and measured, if desired. Using the scattered lightdetectors to measure scattered light at two angle ranges, and at one ormore wavelengths near absorption resonance for the target compound, onemay estimate both particle size and concentration. This may be doneeither through Mie scattering models which iteratively calculate thesequantities by achieving best fits to the data (lookup tables may begenerated in advance in high-throughput systems), or particles may beclassified empirically, either using independent measurements, or bytweaking parameters until known statistical populations are wellseparated in the output of the system. For example, in a system wheresperm cells are being separated by DNA content in order to select X- orY-carrying populations, it is known that roughly 50% of the samplecarries each. Consequently, the outputs of the detectors in the system(at different angles, wavelengths) may be weighted to achieve separationbetween two populations, without reference to a quantitative model todescribe scattering.

In a system where QCL sources are tunable, the measurement wavelengthsmay be optimized empirically as well. In the case ofapplication-specific tools, this may be done in the product developmentstage. In the case of more flexible instruments, or where the analyte(particles, and medium) may vary substantially, the sources may betunable in operation, and may either sweep through the course of ameasurement, or find an optimal measurement wavelength with a subset ofparticles and then perform the main measurement or sorting operation.The generalized function of the scattering-based system is describedhere: one or more frequencies at resonant and optionally non-resonantbands are used, and one or more angle ranges of scattered light aremeasured. The resulting signals may be used to calculate particle volumeand one or more chemical concentrations (or, in some cases, molecularconformations/configurations, which also cause changes inabsorption/refractive index profiles in the mid-IR).

The present invention may be applied to a wide range of applicationswhere infrared vibrational absorption resonances are an indicator ofinterest. One application may be in measurements of biological cells.The label-free measurement of biochemical content in cells is of stronginterest. The present invention has the potential to significantlyimprove accuracy of systems previously described by the inventor. Inaddition, it has the potential to simultaneously measure cellular orcellular component (i.e. nuclear) dimensions and chemical contents. Withembodiments of this disclosure, effective volume and concentration formultiple cellular components could be measured simultaneously usingmultiple wavelengths. For each cellular constituent (protein, DNA,lipid, etc.) a concentration and a spatial distribution figure could becalculated using resonant absorption and/or scattering signals atresonant peaks for each. For example, the invention may be applied todiagnostic applications, such as applications where a large number ofcells are measured in a high-throughput cytometry tool. Cellulardimensions are measured using scattering, and contents such as DNA, RNA,proteins, and/or metabolic products are measured using resonant IRabsorption. Cell population statistics are accumulated and outliersidentified in order to detect abnormalities to diseases. For example, atool which detects malaria from a raw blood sample could isolate DNAmeasurements from red blood cells, where DNA readings over a thresholdcould indicate presence of parasites (sizing capability of the systemused to separate readings from red blood cells from other bloodcomponents). In another example, a system based on the presentdisclosure could refine samples from circulating tumor cell (CTC)capture devices; cells may be characterized by DNA and other content aswell as size.

In another example, embodiments of the disclosure may be applied toassisted reproductive applications. A system based on the presentinvention could accurately determine the DNA content of sperm cells forhuman or animal applications, even as the nuclear volume of the cellsvaries within a patient sample. The ability to measure DNA level allowsseparation of X- and Y-bearing sperm cell for gender selectionapplications. Similar systems could screen out cells with chromosomalabnormalities, or abnormal DNA packing configurations for in vitrofertilization applications. The invention could also be applied toscoring of embryos, where chemical content and byproducts is known tocorrelate with viability, and label-free, non-ionizing radiation-basedtechniques would be highly preferable. The present invention couldcharacterize chemical content of an embryo while compensating forscattering effects, or indeed harnessing resonant scattering to producta more accurate score.

In another example, embodiments of the disclosure may be applied toregenerative medicine. The present invention may be used to refine cellpopulations by both size/shape and chemical content in order to separatepluripotent stem cells from mixtures of cells, without attaching labelsor interrogating the cells with potentially damaging radiation. Fordifferentiated cells generated from stem cells, the system may beapplied to remove any residual pluripotent stem cells before insertioninto patient tissues, in order to prevent tumor formation bynon-differentiated cells.

In another example, embodiments of the disclosure may be applied toindustrial biology. The present invention may be used to characterizeand/or sort cells for industrial processes at high speed. For example,it may be used to refine a culture of cells that produces a high numberof lipids or other products under certain conditions. Such measurementsof cellular products could be done by isolating cells in droplets whichare in turn manipulated in an oil medium as an emulsion.

In another example, embodiments of the disclosure may be applied tocontamination monitoring. Water and other substances may be monitoredfor infectious diseases without pre-processing using the presentinvention. Some filtering for particle/cell size may be performed on theinput, and then particles flowed through a measurement system. Thepresent invention's ability to characterize cell size, generalrefractive index, and resonant absorption/scattering for particularcompounds may allow it to identify specific pathogens. Where pathogensare airborne, the system may capture a sample on an open liquid surface,then flow the liquid through an analysis system. For solid samples suchas food, well-known processes for blending the sample, then filteringthem, may be used to separate potential pathogens before flowing themthrough a system based on the present invention.

Another application may be measurement of emulsions. In many cases it isuseful to measure droplets within an emulsion. This can facilitatehandling in some cases. In other cases the sample being measured isinherently an emulsion. In a droplet-based measurement system, waterdroplets suspended in oil are used to manipulate cells and discretevolumes of chemicals in a microfluidic system. The present inventioncould be used in conjunction with such systems in order to measurechemical concentrations within droplets optically, and at the same timepotentially adjust for the size of droplet. In some cases, the dropletsize measurement may be done at shorter wavelengths. In other cases, acombined measurement around mid-IR resonances is helpful to measureoverall chemical content. In some cases the analyte of interest is anemulson of chemicals. The present invention can serve to characterizesuch mixtures both by chemical content and by particle size. This may beuseful in a number of industrial and food processes.

Another application is in the measurement of solid particles insuspension. With the present invention, it may in some cases bedesirable to build a system that places solid particles into a liquidsuspension in order to characterize them. For example a system mayinclude a capability that collects a solid sample from a surface throughscraping, milling or vacuuming, then introduces these samples into aliquid medium, then runs these samples through one or more size filtersto select particles that are appropriate for measurement and excludeunwanted substances (including, potentially, size-sorting microfluidicdevices such as those demonstrated by Robert Austin at PrincetonUniversity), then runs the particles through the an optical measurementsystem based on the present invention. The liquid medium may be tuned tohave specific refractive index relationship to the particles ofinterest, so that it in effect becomes a “reference” in the system. Evenif the particles are very dense and have high optical absorption atresonant wavelengths, the present scattering-compensated/enhancedmeasurements system may provide volumetrically-compensated chemicalcomposition information, or both volume and chemical compositioninformation. Such systems may be of interest in applications wherediffuse reflective or ATR-type spectroscopy are currently used, butsignals are insufficiently accurate or sensitive. This may include caseswhere there is a very thin layer of interest on an object (the describedsystem serves to consolidate the layer into a flow), or where theunderlying substances interfere with measurement.

Emulsion-based microsystems for handling of small volumes of analyte orbiological materials, including cells, are an area with growingactivity. One common challenge in such systems is measurement of dropletor flow contents, where individual volumes are tiny. It would be highlydesirable to be able to measure the contents of such an emulsionnon-invasively, without adding additional chemicals that may disturb thechemical reaction or cellular metabolism, and without radiation thatcould disturb or harm the contents of the droplets, cells, emulsions,etc.

The unique properties that a QCL-based system brings to an emulsion ordroplet-based fluidic system include the ability to provide highspectral power density at specific wavelengths in the mid-IR and THzregime corresponding to molecular bond vibrations; and the ability tofocus this light very tightly and efficiently onto a small spot for thepurpose of interrogating individual particles, droplets, biologicalcells, etc. with micron resolution, thereby creating the ability tomeasure these at a high rate while resolving them individually.Correspondingly, the low etendue of QCL light sources allows light to bevery well and efficiently collimated in the case where it is desirableto have the angle distribution of the illuminating source to be verynarrow.

Unlike most Raman spectroscopy systems that probe the same molecularvibrations, mid-IR systems have very strong interaction with the targetmolecules. In addition, mid-IR light, including light from QCLs, has theadvantage that it is very low energy (per photon), minimizing the chanceof photon damage. Finally, mid-IR light has the advantage of longwavelength vs. UV/Visible/NIR and Raman measurements; this longwavelength reduces scattering effects and makes them more easilymanageable in a measurement system.

The fluidics may be fabricated using materials appropriate for mid-IRlight transmission and designed optically to avoid fringing andresonances at the wavelengths employed.

A number of configurations are described below. Any of theconfigurations, techniques, architectures previously described usingQCLs to interrogate particles, cells, droplets and sub-flows in fluidsare applicable to these.

One additional optical method that may be applied to the previousconfigurations disclosed herein adds polarization as a sensing modalityto the QCL-based interrogation of these particles in a flow. Ifmolecules being interrogated by mid-IR vibrational spectroscopy arearranged in specific manners within the particle being measured—forexample, DNA in helical configuration—the measured absorption at theabsorption band of the molecules will depend on the polarization of themid-IR light. One may alternately polarize the light in left and rightcircular polarization and measure the differential. The observeddifferential, so called vibrational circular dichroism (VCD) may providea particularly sensitive measurement of chiral or helical molecules,and/or provide information about the folding or configuration of aparticular molecule within the analyzed particle/cell/droplet.

QCL-VCD interrogation of particles in a fluid may be combined with othertechniques described herein, such as scattering, or use of mid-IR activelabels/dyes, to measure certain types of target molecules.

One example of a target molecule measurement is DNA measurement. DNA isa helical molecule and therefore exhibits polarization-dependentabsorption at resonant absorption bands of its constituents (phosphateand deoxyribose components on its backbone). This property may be usedto separate a DNA absorption signature from other analytes whenmeasuring using one or more QCL wavelengths. It may also be used todetermine, with high accuracy, the folding or packing state of DNAwithin a cell nucleus.

FIG. 50a shows a simple flow architecture where the fluid to be measuredflows through a channel, where it is interrogated using a mid-IR or THzQCL-derived beam (shown as dotted ellipse). The transmission at one ormore wavelengths through the channel is measured to determine chemicalconcentrations within the flow.

FIG. 50b shows a fluid-within-fluid flow, also within a channelstructure or manifold. At small scales, as is well known, fluids tend toremain in a laminar (non-turbulent/non-mixing). This enables a “core”flow of analyte to remain centered and unmixed with a “sheath” flow, asis shown here. This method of presenting an analyte (which may be aliquid, liquid with solid particles and/or biological matter, liquidwith dissolved gases, emulsion, or suspension) gives some additionalpossibilities for QCL-based measurements. First, it eliminates potentialartifacts arising from the laser beam (dotted ellipse) crossing the edgeof the fluid channel. Second, in this configuration it is morestraightforward to make scattering-based measurements that accentuatedifferences between the core and sheath fluids. Refractive index (bothreal and imaginary) differences between the sheath and core flows willresult in optical interference effects (often described as Miescattering for particles), effectively changing the angle of some of thelight transmitted through the flow. As discussed earlier, in the mid-IRregime addressable by QCLs, there are relatively narrow, resonantrefractive index variations (dispersion) around absorption peakscharacteristic of molecular bond vibrations. These may be exploited,with QCL-derived illumination whose angle is well controlled, to getvery sensitive concentration measurements within the fluid flow.Depending on the concentration of a particular molecule within the coreflow, there will be characteristic variations in refractive index—andtherefore in observed scattering/diffraction intensity and angle—as afunction of mid-IR wavelength.

The flow shown in FIG. 50b may simply be two fluids (core and sheath)flowing laminarly, but not otherwise separated. Alternatively, the flowmay be an emulsion, where the two fluids naturally remain separated. Forexample, the sheath fluid could be oil, whereas the core flow could bean aqueous solution. Several measurements could then be performed usingthe QCL-based architectures previously disclosed: the characteristics ofthe core flow (dimension, base index) could be measured at onewavelength where the molecule of interest is not resonant; then otherwavelengths could be used to measure specific chemical concentrations ofinterest, using either direct transmission/absorption measurements, orscattering-based measurements. Knowledge of the core flow diameterderived from the initial measurement may then be used to compensate thetarget-specific signal, for example accurately calculating concentrationof the chemical within the core flow. Multiple angles (directtransmission, and multiple scatter angles) may be used to calculatedimensions and concentrations.

With this as well as other architectures disclosed herein, multipleconfigurations for measuring scattering over multiples angles may beused. Discrete detectors capturing light scattered at different anglesmay be used, for example. In another configuration, a mid-IR focal planearray may be used to simultaneously measure light emerging at differentangles from the measured fluid/cell/particle. Alternatively, mirrorsystems may be used to sample portions of the output beam. Mirror arrayssuch as the Texas Instruments digital micromirrors may be used to sampleportions of the emerging beam and relay them to a single detector (suchas a high-speed MCT). Rotating mirrors may be used to scan the emergingbeam over a detector with an aperture, either along 1 or 2 axes (usefulwhere potentially asymmetric particles/droplets are being measured).

These architectures, with two or more QCL wavelengths, one or more ofwhich corresponds to wavelengths where a substance of interest has aresonant dispersion feature, may be used with multiple architecturesthat present particles, cells, fluids, droplets and the like to themid-IR beams. For example, this QCL-based resonant Mie scatteringarchitecture may be used with cells that have been mounted on a mid-IRtransparent substrate (or mid-IR reflective substrate).

FIG. 50c shows an example of a flow where the core has been broken intodroplets. These droplets may be emulsed in the sheath flow. The dropletspass through the QCL measurement beam (dotted line) and are measuredusing direct transmission and/or scattered light detection.

All of these architectures based on mid-IR QCL light may be combinedwith other measurement techniques, including optical. These include butare not limited to scattering measurements, fluorescence measurements,and others previously disclosed.

FIG. 51a shows an example of scattering efficiency (Qs) of a volume suchas a droplet, as a function of wavelength (lambda). Assuming relativelyconstant refractive index for the volume as well as the surroundingmedium, scattering drops as a function of wavelength.

FIG. 51b shows scattering efficiency as a function of wavelength where achemical within the droplet has a vibrational absorption band in thewavelength range being displayed. A local increase in absorptionnecessarily corresponds (as can be determined from the Kramers-Kronigrelation) to a local resonant variation in real refractive index. Thisvariation in real index results in a local (wavelength) perturbation ofthe optical interference pattern (scattering) arising as the mid-IR beampasses through the droplet and surrounding medium. The term “droplet”here is used to mean droplet, cell, particle within another liquid,including cases where the droplet is in an emulsion.

It should be noted that several mid-IR (or other) wavelengths may beused to accurately determine chemical concentration within such asystem. The resonant dispersion (local index variation around theabsorption band) measurements are done using mid-IR QCLs. Otherwavelengths in non-resonant regimes may be used to measure the overalldroplet (size, shape, orientation) by scattering. For this, visible,NIR, or mid-IR wavelengths may be used. Visible wavelengths are alreadyused to assess size and shape in systems such as blood count toolstoday. They, however, do not have the capability of doingchemistry-specific measurements enabled by the mid-IR resonantscattering architecture disclosed herein.

FIG. 52a shows a fluidic configuration where a droplet or flow isconfined within a 2D channel or manifold, such as those found in manymicrofluidic chip architectures. Mid-IR light from one or more QCLspasses through the droplet and the surrounding medium. Depending on thewavelength and chemical concentrations within the droplet andsurrounding medium, light is absorbed and/or scattered.

FIG. 52b shows a fluidic system where droplets or a core flow iscentered within a larger, 3D core flow. This is more typical ofconventional flow cytometer cuvettes, for instance. The QCL-basedmeasurements described previously are then made of the droplet or flow,based on directly transmitted or scattered mid-IR light.

The fluidic architectures shown in cross section in FIGS. 52a-b may beused in cases where the “droplets” or “flows” are in emulsion; in otherwords, where the droplets shown here do not mix with the surroundingflow, even when stationary.

FIGS. 53a-c show a representative example of a system measuring dropletsor flows (within a sheath flow, which is not shown) using QCL-originatedmid-IR beams. They are used to illustrate scattering as a function ofthe flow/droplet content, and mid-IR wavelength, as described under FIG.53.

FIG. 53a shows the system with the sheath flow only, or a baselinestate. All mid-IR light is focused onto a single point, corresponding totraversal of the sheath fluid without angular perturbation resultingfrom refractive index differences.

FIG. 53b shows the system as a droplet or core flow are introduced. Thedroplet will cause some differential absorption of thedirectly-transmitted mid-IR light (this could be in some cases benegative, where the droplet absorbs less than the sheath flow). As afunction of refractive index and wavelength, the droplet will alsodiffract some of the mid-IR light so it exits the flow off-axis. Whenimaged onto a plane, this light will appear away from the central spot.As described earlier, this pattern will be wavelength-dependent, andaround certain resonant absorption wavelengths, will have strongerwavelength dependence. A combination of the directly-transmitted(central) signal and scattered signal as a function of mid-IR wavelengthand scattering angle (position on the plane) may then be used toestimate both droplet dimensions and specific chemical concentrationswithin the droplet. The droplet, here, could be a continuous flow ofliquid (viewed in cross-section), either in laminar flow or emulsed in asheath fluid; it could be a single droplet; it could be a biologicalcell; a solid particle; or other form of spectrally-measurable object ina liquid flow, as described earlier.

FIG. 53c shows the same droplet or flow, now with another object insideof it. For example, this could be a cell, a dense nucleus or otherorganelle. It could be a droplet in an emulsion containing a single cell(or multiple cells). The system may be used again, as described above,to characterize the droplet, and then to characterize the particlewithin the droplet using the same techniques, often using differentmid-IR wavelengths that correspond to molecular bond vibrations ofinterest within the contained particle. Again, this will result inabsorption of the QCL-originated light passing through the containedparticle, and scattering of the light based on refractive index at thosewavelengths, which may show resonant dispersion around characteristicabsorption lines.

In this architecture, a tunable QCL could be used to interrogate aseries of wavelengths corresponding to non-resonant (reference)wavelengths, then resonant wavelengths corresponding to the dropletfluid and particle fluid, respectively.

In some cases, the droplets may accumulate chemical species as a resultof chemical reactions that have occurred inside of them, or biologicalprocesses occurring in a cell contained within them (possiblystimulated/inhibited with additives to the droplet).

FIG. 54a shows a case where a biological cell (inner circle) iscontained within a droplet, which is contained within an emulsion. Anadvantage of such a system, described in detail in Boehm et al, is thatthe droplet acts as an independent volume, not mixing with thesurrounding (transport/sheath) liquid. As a result, chemical reactionscan be performed at a tiny scale, and their results measured (assumingan appropriate measurement technique, such as ours). Alternatively, asis shown in this representative example, a cell may be incubated in thedroplet with nutrients, drugs, toxins and/or other substances, inparallel with thousands or millions of other cells in a microfluidicsystem. At a fixed time, the present invention may be used tointerrogate the droplet using QCL-derived light. At this point, thecontents of the cell may be measured directly using vibrationalspectroscopy techniques described herein. However, it may also be ofinterest to measure the contents of the droplets, which will contain themetabolic byproducts of the cell, accumulated in the course ofincubation. The byproducts and their concentration may be a powerfulindicator of cell function. The droplet contents may be measured usingthe QCL-based techniques described herein. In some cases, only thecontents of the emulsed droplet itself will be of interest, and thetechniques described above may be used to “substract out” the opticalsignature of the cell from the transmitted and/or scattered mid-IRlight.

FIG. 54b shows a different technique, illustrated here through the useof a droplet in an emulsion. Here the droplet is formed, containing acell, but also containing a dye or label with a known and preferablydistinct mid-IR vibrational signature. As with fluorescent dyes/labels,and emerging quantum dot based tags, it is functionalized in order tobind to or localize in particular portions of the cell, possiblydepending on the cell phenotype, surface antibodies, etc.

The advantage of using a mid-IR dye/label, with readout using QCL-basedmid-IR interrogation (absorption and scattering) are multiple: (1) ahigh degree of chemical flexibility, since virtually all molecules havemid-IR fingerprints; (2) the potential for a large number of labelssimultaneously present and measured, because of the complex fingerprintsachievable; (3) well-behaved scattering at mid-IR wavelengths, at thescale of cells and organelles. Moreover, scattering, as has beendescribed, can be measured in a very chemical- and size-specific manner.As a result, it is possible to measure chemical concentration andassociated structural information using resonant Mie scattering andQCL-originated beams with well-controlled angular incidence.

In this example, the droplet is filled with a mid-IR label, which overthe course of incubation, binds to specific structures on the containedcell. By interrogating the droplet using QCL beam(s) after thisincubation, one may determine how much of the label has beenconcentrated on the surface of, in the nucleus of, or within anotherorganelle of the cell.

This technique of labeling using mid-IR active dyes/labels/particles maybe applied with QCL-based vibrational absorption/scatteringarchitectures previously disclosed by the inventor. It is potentially astrong complement to the label-free techniques previously disclosed, andto UV/visible/NIR techniques in broad industry use today.

FIG. 55 shows an example of the present invention used in adroplet-based system where droplets contain biological cells. Thedroplets may also be seeded with growth media, nutrients, drugs, etc. Ingeneral it may be desirable to have a set number of cells (often one)per droplet; generally the number of cells will follow a Poissondistribution as described by Boehm et al. The cells may be incubated fora fixed period at specific conditions, and then run into a measurementvolume as shown here, where microfluidic channels guide individualdroplets into a flow channel with a sheath flow that centers thedroplets.

Alternatively, various focusing techniques, including acoustic, optical,and mechanical may be used to center the flow of droplets and space themappropriately for measurement, and in some cases, subsequent sorting.

In this configuration, multiple wavelengths of QCL (or, tunable QCL(s))may be used in conjunction with direct transmission and/or scatteringdetectors in order to determine any one or combination of the following:(1) how many cells are present within a particular droplet, potentiallyeliminating from consideration those with the wrong number of cells, orcompensating remaining measurements appropriately; (2) gatheringspectral/scattering information regarding the cells to ascertain theircontents or phenotype; (3) determine droplet volume; and (4) measurechemical contents of the droplets to measure, for example, the metabolicproducts of the cell(s) contained within. The resulting measurementscould include anything from simple characterization of what type ofcell(s) are contained within droplets, of the purpose of sorting,diagnostics or statistical measurements; what the contents of the cellsare, including contents such as DNA, RNA, protein, sugars, lipids,metabolic byproducts, etc.; what chemicals are produced by the cell. Themeasurements may be recorded, or used to sort droplets in real time.

In other configurations, the droplets may be immobilized in “pots” wherethey may be observed over time. In this manner, time series (or at leastbefore/after) measurements may be made of droplets maintained orprocessed under specific conditions. The array of pots may either betranslated across the QCL beam for one-by-one measurement, or the potsmay be unloaded sequentially past a QCL measurement point.

FIG. 56 shows another configuration where a solid sample with verysparse (potential) particles of interest is to be analyzed. The processof analyzing a bulk sample for trace chemical or biological componentsis often difficult because signal is low when the entire sample ismeasured, or highly variable if small points are measured (for example,by ATR prism using FTIR).

According to the present invention, the solid may be milled into a finepowder (if it is not already in powder form), and then introduced intoan appropriate liquid. Depending on the material being analyzed, thiscould be water, alcohol, oils or other liquids. The particles may thenbe sorted or filtered to achieve uniform size, potentially through theuse of microfluidic structures. The particles are then incorporated intodroplets in an emulsion. These droplets may, in some cases, contain notonly the particles but also additive chemicals or tags that attachto/react with the molecules of interest in the solid, and may producebyproducts detectable by mid-IR vibration spectroscopy.

The droplets are then interrogated, possibly after some reaction time,using one or more QCL beams, in order to determine the content of thesolid particle, by direct measurement of the particle, by measurement ofcomplexes formed between additives and the particle, or byproducts ofreactions between the particle and additives.

Particles here could include minerals of interest, trace pollutants orcontaminants, explosives or chemical/biological weapons traces, ormicrobes including food contaminants.

FIGS. 57a-c show flow and particle configurations that could be used inthe present invention to enhance resonant optical interferencemeasurements in the mid-IR. Here, rather than a single particle, dropletor stream producing an interference (scattering) pattern whenQCL-originated mid-IR beams hits it, multiple parallel particles orstreams are used to produce a periodic “grating” in order to enhancescattering effects and increase signal-to-noise for specific chemicalbond detection.

FIG. 57a shows parallel laminar flows which may be formed initially byrelatively large microfluidic nozzles, and then narrowed to theappropriate dimensions, to form a liquid diffraction grating which mayserve to measure the liquid content with very high specificity andaccuracy in the mid-IR. In such a configuration, the diffraction anglesof light imparted by the grating as light passes through it(perpendicular to the page) depends on the phase difference imparted bythe alternating fingers, and therefore on the relative refractive indexbetween the flows. As noted, where there are chemicals present in oneset of flows that have resonant absorption bands in the mid-IR, thereare associated refractive index fluctuations (resonant dispersion) nearthe same wavelengths, and two or more QCL-based measurements atwavelengths in or around these resonant features made with detectorsmeasuring off-angle transmission can result in precise chemicalmeasurements. Both resonant and non-resonant measurements may be made toaccurately determine concentration and compensate for size and operatingenvironment effects.

FIG. 57b shows a series of droplets passing through an asymmetric beamat regular intervals to similarly form a liquid diffraction grating.

FIG. 57c shows an architecture where particles, cells or bubbles arefocused using acoustic, fluidic or optical means into parallel streamsfor interrogation by QCL beam(s) according to the present invention.

FIG. 58 shows one method by which cells or particles could be measuredand sorted using QCL-based vibration spectroscopic techniques describedherein, and droplet emulsions described by Boehm et al. In this example,a double emulsion is used, where cells are contained in a water-baseddroplet, which is encased in an oil “shell” (a double, or tripleemulsion), which itself is suspended in a water-based medium. Accordingto methods described above in the invention, QCL based beam(s) are usedto interrogate the cell and/or surrounding liquid that (for example)contains metabolic byproducts from the cell. Depending on the outcome ofthe measurement, the system may break open the oil shell and release thecontained cell. This release step may be performed using one of a numberof means, including but not limited to: acoustic forces that break upthe droplet, optical pulses which force a hole into the shell (includingbut not limited to mid-IR radiation which may target specific absorptionlines for the oil), or mechanical. After release, cells still in oilcases may be separated passively by appropriate microfluidic structuresfrom cells which have been released from their shells.

The methods and systems described herein may be deployed in part or inwhole through a machine that executes computer software, program codes,and/or instructions on a processor. The processor may be part of aserver, cloud server, client, network infrastructure, mobile computingplatform, stationary computing platform, or other computing platform. Aprocessor may be any kind of computational or processing device capableof executing program instructions, codes, binary instructions and thelike. The processor may be or include a signal processor, digitalprocessor, embedded processor, microprocessor or any variant such as aco-processor (math co-processor, graphic co-processor, communicationco-processor and the like) and the like that may directly or indirectlyfacilitate execution of program code or program instructions storedthereon. In addition, the processor may enable execution of multipleprograms, threads, and codes. The threads may be executed simultaneouslyto enhance the performance of the processor and to facilitatesimultaneous operations of the application. By way of implementation,methods, program codes, program instructions and the like describedherein may be implemented in one or more thread. The thread may spawnother threads that may have assigned priorities associated with them;the processor may execute these threads based on priority or any otherorder based on instructions provided in the program code. The processormay include memory that stores methods, codes, instructions and programsas described herein and elsewhere. The processor may access a storagemedium through an interface that may store methods, codes, andinstructions as described herein and elsewhere. The storage mediumassociated with the processor for storing methods, programs, codes,program instructions or other type of instructions capable of beingexecuted by the computing or processing device may include but may notbe limited to one or more of a CD-ROM, DVD, memory, hard disk, flashdrive, RAM, ROM, cache and the like.

A processor may include one or more cores that may enhance speed andperformance of a multiprocessor. In embodiments, the process may be adual core processor, quad core processors, other chip-levelmultiprocessor and the like that combine two or more independent cores(called a die).

The methods and systems described herein may be deployed in part or inwhole through a machine that executes computer software on a server,client, firewall, gateway, hub, router, or other such computer and/ornetworking hardware. The software program may be associated with aserver that may include a file server, print server, domain server,internet server, intranet server and other variants such as secondaryserver, host server, distributed server and the like. The server mayinclude one or more of memories, processors, computer readable media,storage media, ports (physical and virtual), communication devices, andinterfaces capable of accessing other servers, clients, machines, anddevices through a wired or a wireless medium, and the like. The methods,programs or codes as described herein and elsewhere may be executed bythe server. In addition, other devices required for execution of methodsas described in this application may be considered as a part of theinfrastructure associated with the server.

The server may provide an interface to other devices including, withoutlimitation, clients, other servers, printers, database servers, printservers, file servers, communication servers, distributed servers,social networks, and the like. Additionally, this coupling and/orconnection may facilitate remote execution of program across thenetwork. The networking of some or all of these devices may facilitateparallel processing of a program or method at one or more locationwithout deviating from the scope of the invention. In addition, any ofthe devices attached to the server through an interface may include atleast one storage medium capable of storing methods, programs, codeand/or instructions. A central repository may provide programinstructions to be executed on different devices. In thisimplementation, the remote repository may act as a storage medium forprogram code, instructions, and programs.

The software program may be associated with a client that may include afile client, print client, domain client, internet client, intranetclient and other variants such as secondary client, host client,distributed client and the like. The client may include one or more ofmemories, processors, computer readable media, storage media, ports(physical and virtual), communication devices, and interfaces capable ofaccessing other clients, servers, machines, and devices through a wiredor a wireless medium, and the like. The methods, programs or codes asdescribed herein and elsewhere may be executed by the client. Inaddition, other devices required for execution of methods as describedin this application may be considered as a part of the infrastructureassociated with the client.

The client may provide an interface to other devices including, withoutlimitation, servers, cloud servers, other clients, printers, databaseservers, print servers, file servers, communication servers, distributedservers and the like. Additionally, this coupling and/or connection mayfacilitate remote execution of program across the network. Thenetworking of some or all of these devices may facilitate parallelprocessing of a program or method at one or more location withoutdeviating from the scope of the invention. In addition, any of thedevices attached to the client through an interface may include at leastone storage medium capable of storing methods, programs, applications,code and/or instructions. A central repository may provide programinstructions to be executed on different devices. In thisimplementation, the remote repository may act as a storage medium forprogram code, instructions, and programs.

The methods and systems described herein may be deployed in part or inwhole through network infrastructures. The network infrastructure mayinclude elements such as computing devices, servers, cloud servers,routers, hubs, firewalls, clients, personal computers, communicationdevices, routing devices and other active and passive devices, modulesand/or components as known in the art. The computing and/ornon-computing device(s) associated with the network infrastructure mayinclude, apart from other components, a storage medium such as flashmemory, buffer, stack, RAM, ROM and the like. The processes, methods,program codes, instructions described herein and elsewhere may beexecuted by one or more of the network infrastructural elements.

The methods, program codes, and instructions described herein andelsewhere may be implemented on a cellular network having multiplecells. The cellular network may either be frequency division multipleaccess (FDMA) network or code division multiple access (CDMA) network.The cellular network may include mobile devices, cell sites, basestations, repeaters, antennas, towers, and the like. The cell networkmay be a GSM, GPRS, 3G, EVDO, mesh, or other networks types.

The methods, programs codes, and instructions described herein andelsewhere may be implemented on or through mobile devices. The mobiledevices may include navigation devices, cell phones, mobile phones,mobile personal digital assistants, laptops, palmtops, netbooks, pagers,electronic books readers, music players and the like. These devices mayinclude, apart from other components, a storage medium such as a flashmemory, buffer, RAM, ROM and one or more computing devices. Thecomputing devices associated with mobile devices may be enabled toexecute program codes, methods, and instructions stored thereon.Alternatively, the mobile devices may be configured to executeinstructions in collaboration with other devices. The mobile devices maycommunicate with base stations interfaced with servers and configured toexecute program codes. The mobile devices may communicate on a peer topeer network, mesh network, or other communications network. The programcode may be stored on the storage medium associated with the server andexecuted by a computing device embedded within the server. The basestation may include a computing device and a storage medium. The storagedevice may store program codes and instructions executed by thecomputing devices associated with the base station.

The computer software, program codes, and/or instructions may be storedand/or accessed on machine readable media that may include: computercomponents, devices, and recording media that retain digital data usedfor computing for some interval of time; semiconductor storage known asrandom access memory (RAM); mass storage typically for more permanentstorage, such as optical discs, forms of magnetic storage like harddisks, tapes, drums, cards and other types; processor registers, cachememory, volatile memory, non-volatile memory; optical storage such asCD, DVD; removable media such as flash memory (e.g. USB sticks or keys),floppy disks, magnetic tape, paper tape, punch cards, standalone RAMdisks, Zip drives, removable mass storage, off-line, and the like; othercomputer memory such as dynamic memory, static memory, read/writestorage, mutable storage, read only, random access, sequential access,location addressable, file addressable, content addressable, networkattached storage, storage area network, bar codes, magnetic ink, and thelike.

The methods and systems described herein may transform physical and/oror intangible items from one state to another. The methods and systemsdescribed herein may also transform data representing physical and/orintangible items from one state to another.

The elements described and depicted herein, including in flow charts andblock diagrams throughout the figures, imply logical boundaries betweenthe elements. However, according to software or hardware engineeringpractices, the depicted elements and the functions thereof may beimplemented on machines through computer executable media having aprocessor capable of executing program instructions stored thereon as amonolithic software structure, as standalone software modules, or asmodules that employ external routines, code, services, and so forth, orany combination of these, and all such implementations may be within thescope of the present disclosure. Examples of such machines may include,but may not be limited to, personal digital assistants, laptops,personal computers, mobile phones, other handheld computing devices,medical equipment, wired or wireless communication devices, transducers,chips, calculators, satellites, tablet PCs, electronic books, gadgets,electronic devices, devices having artificial intelligence, computingdevices, networking equipments, servers, routers and the like.Furthermore, the elements depicted in the flow chart and block diagramsor any other logical component may be implemented on a machine capableof executing program instructions. Thus, while the foregoing drawingsand descriptions set forth functional aspects of the disclosed systems,no particular arrangement of software for implementing these functionalaspects should be inferred from these descriptions unless explicitlystated or otherwise clear from the context. Similarly, it will beappreciated that the various steps identified and described above may bevaried, and that the order of steps may be adapted to particularapplications of the techniques disclosed herein. All such variations andmodifications are intended to fall within the scope of this disclosure.As such, the depiction and/or description of an order for various stepsshould not be understood to require a particular order of execution forthose steps, unless required by a particular application, or explicitlystated or otherwise clear from the context.

The methods and/or processes described above, and steps thereof, may berealized in hardware, software or any combination of hardware andsoftware suitable for a particular application. The hardware may includea general purpose computer and/or dedicated computing device or specificcomputing device or particular aspect or component of a specificcomputing device. The processes may be realized in one or moremicroprocessors, microcontrollers, embedded microcontrollers,programmable digital signal processors or other programmable device,along with internal and/or external memory. The processes may also, orinstead, be embodied in an application specific integrated circuit, aprogrammable gate array, programmable array logic, or any other deviceor combination of devices that may be configured to process electronicsignals. It will further be appreciated that one or more of theprocesses may be realized as a computer executable code capable of beingexecuted on a machine readable medium.

The computer executable code may be created using a structuredprogramming language such as C, an object oriented programming languagesuch as C++, or any other high-level or low-level programming language(including assembly languages, hardware description languages, anddatabase programming languages and technologies) that may be stored,compiled or interpreted to run on one of the above devices, as well asheterogeneous combinations of processors, processor architectures, orcombinations of different hardware and software, or any other machinecapable of executing program instructions.

Thus, in one aspect, each method described above and combinationsthereof may be embodied in computer executable code that, when executingon one or more computing devices, performs the steps thereof. In anotheraspect, the methods may be embodied in systems that perform the stepsthereof, and may be distributed across devices in a number of ways, orall of the functionality may be integrated into a dedicated, standalonedevice or other hardware. In another aspect, the means for performingthe steps associated with the processes described above may include anyof the hardware and/or software described above. All such permutationsand combinations are intended to fall within the scope of the presentdisclosure.

While the invention has been disclosed in connection with the preferredembodiments shown and described in detail, various modifications andimprovements thereon will become readily apparent to those skilled inthe art. Accordingly, the spirit and scope of the present invention isnot to be limited by the foregoing examples, but is to be understood inthe broadest sense allowable by law.

All documents referenced herein are hereby incorporated by reference.

1-4. (canceled)
 5. A system for measuring a fluid comprising: a channelwith a measurement volume into which two fluids are introduced; at leastone laser source configured to emit at least a light at one or morewavelengths towards at least one of the two fluids in an illuminationregion, wherein after interacting with the at least one of the twofluids comprising any respective constituent, the light undergoes adetectable change based on a property of the least one of the two fluidsinclusive of the any respective constituent; a detector configured todetect the detectable change; and at least one processor configured toanalyze the detectable change and determine the property of the at leastone of the two fluids inclusive of the any respective constituent. 6.The system of claim 5, wherein the two fluids are in physical contactwith each other and are substantially separated by at least one boundarybetween the two fluids; and wherein the at least one laser illuminatesthe two fluids at the at least one boundary in the illumination region.7. The system of claim 6, wherein the two fluids are in laminar flowwith respect to each other and the at least one boundary between the twofluids is parallel to the laminar flow; or wherein the two fluids arenot in laminar flow with respect to each other and the at least oneboundary between the fluids is perpendicular to the flow of the twofluids.
 8. The system of claim 7, wherein a first fluid of the twofluids is a droplet encompassed within a second fluid of the two fluids.9. The system of claim 6, wherein the detector is configured to detecttemporally the detectable change of the light after the light impactsthe two fluids as the at least one boundary passes substantiallyperpendicularly to the flow in the illumination region.
 10. The systemof claim 6, wherein the detector is configured to detect spatially thedetectable change of the light after the light interacts with the twofluids as the at least one boundary between the fluids passessubstantially parallel to the flow in the illumination region.
 11. Thesystem of claim 5, wherein the channel comprises a 2D microfluidicchannel and wherein a first fluid of the two fluids is confined withinthe 2D microfluidic channel; or wherein the channel comprises a 3D coreand wherein the first fluid of the two fluids is centered within the 3Dcore.
 12. The system of claim 5, wherein the at least one laser sourceis selected from the group consisting of an infrared laser source, a THzlaser source, and a combination thereof.
 13. The system of claim 12,wherein the infrared laser source or the THz laser source is a quantumcascade laser.
 14. The system of claim 5, wherein the at least one lasersource is further configured to emit the at least one light at awavelength corresponding to or around a resonant absorption or at awavelength corresponding to or near an absorption peak for at least oneof the two fluids inclusive of the any respective constituent.
 15. Thesystem of claim 5, wherein the detectable change in light is selectedfrom the group consisting of a difference in absorption between the twofluids inclusive of the any respective constituents; a difference inrefractive index between the two fluids inclusive of the any respectiveconstituents; a difference in transmission of differently polarizedlight between the two fluids inclusive of the any respectiveconstituents; and a combination thereof.
 16. The system of claim 5,wherein the property resulting in the detectable change comprises achemical property or a physical property of the any respectiveconstituent within one or both of the fluids.
 17. The system of claim16, wherein the physical property resulting in the detectable changecomprises a molecular configuration of the any respective constituentwithin one or both of the fluids.
 18. The system of claim 17, whereinthe molecular configuration comprises a particular shape.
 19. The systemof claim 6, wherein the detectable change based on the property of thefirst fluid inclusive of the any respective constituent is normalizedrelative to the detectable change based on the property of the secondfluid inclusive of the any respective constituent.
 20. The system ofclaim 5, wherein the any respective constituent comprises a solid thatis fractured into particles suspended in liquid; and wherein theparticles are filtered to ensure a particular maximum diameter or shape.21. A method for measuring a liquid comprising introducing two fluidsinto a channel with a measurement volume; emitting at least one light atone or more wavelengths from at least one laser source towards at leastone of the two fluids in an illumination region, wherein afterinteracting with the at least one of the two liquids comprising anyrespective constituent, the light undergoes a detectable change based ona property of the least one of the two liquids inclusive of the anyrespective constituent; detecting the detectable change with a detector;and analyzing the detectable change and determining the property of theat least one of the two liquids inclusive of the any respectiveconstituent with at least one processor.
 22. The method of claim 21,wherein the two fluids are in physical contact with each other and aresubstantially separated by at least one boundary between the two fluids;and wherein the at least one laser illuminates the two fluids at the atleast one boundary in the illumination region.
 23. The method of claim22, wherein the two fluids are in laminar flow with respect to eachother and the at least one boundary between the two fluids is parallelto the laminar flow; or wherein the two fluids are not in laminar flowwith respect to each other and the at least one boundary between thefluids is perpendicular to the flow of the two fluids.
 24. The method ofclaim 23, wherein a first fluid of the two fluids is a dropletencompassed within a second fluid of the two fluids.
 25. The method ofclaim 21, wherein the detector is configured to detect temporally thedetectable change of the light after the light impacts the two fluids asthe at least one boundary passes substantially perpendicularly to theflow in the illumination region.
 26. The method of claim 21, wherein thedetector is configured to detect spatially the detectable change of thelight after the light interacts with the two fluids as the at least oneboundary between the fluids passes substantially parallel to the flow inthe illumination region.
 27. The method of claim 21, wherein the channelcomprises a 2D microfluidic channel and wherein a first fluid of the twofluids is confined within the 2D microfluidic channel; or wherein thechannel comprises a 3D core and wherein the first fluid of the twofluids is centered within the 3D core.
 28. The method of claim 21,wherein the at least one laser source is selected from the groupconsisting of an infrared laser source, a THz laser source, and acombination thereof.
 29. The method of claim 28, wherein the infraredlaser source or the THz laser source is a quantum cascade laser.
 30. Themethod of claim 21, wherein the at least one laser source is furtherconfigured to emit the at least one light at a wavelength correspondingto or around a resonant absorption or at a wavelength corresponding toor near an absorption peak for at least one of the two fluids inclusiveof the any respective constituent.
 31. The method of claim 21, whereinthe detectable change in light is selected from the group consisting ofa difference in absorption between the two fluids inclusive of the anyrespective constituents; a difference in refractive index between thetwo fluids inclusive of the any respective constituents; a difference intransmission of differently polarized light between the two fluidsinclusive of the any respective constituents; and a combination thereof.32. The method of claim 21, wherein the property resulting in thedetectable change comprises a chemical property or a physical propertyof the any respective constituent within one or both of the fluids. 33.The method of claim 32, wherein the physical property resulting in thedetectable change comprises a molecular configuration of the anyrespective constituent within one or both of the fluids.
 34. The methodof claim 33, wherein the molecular configuration comprises a particularshape.
 35. The method of claim 21, wherein the detectable change basedon the property of the first fluid inclusive of the any respectiveconstituent is normalized relative to the detectable change based on theproperty of the second fluid inclusive of the any respectiveconstituent.
 36. The method of claim 21, wherein the any respectiveconstituent comprises a solid that is fractured into particles suspendedin liquid; and wherein the particles are filtered to ensure a particularmaximum diameter or shape.